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Full text of "Nuclear ribosomal DNA sequence analysis in molecular systematics of Pezizales (Ascomycetes)"

NUCLEAR RIBOSOMAL DNA SEQUENCE ANALYSIS IN MOLECULAR 
SYSTEMATICS OF PEZIZALES ( ASCOMYCETES ) 



By 
ESENGUL A. MOMOL 



A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL 
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF 
THE REQUIREMENTS FOR THE DEGREE OF 
DOCTOR OF PHILOSOPHY 

UNIVERSITY OF FLORIDA 

1992 






'WJij'W! 



m OF FLOHIOA LffiRKSl 



ACKNOWLEDGEMENTS 

I would like to express my deepest appreciation and 
gratitude to Dr. James W. Kimbrough, for his guidance, 
support, encouragement and his limitless patience throughout 
this research. I was indeed fortunate to have him as a 
committee chairman. His attitude toward the students, 
encouraging their thoughts and creativity, challenging their 
ideas and most importantly, his constructive criticism make 
him an excellent example of an educator. I wish to extend 
my profound appreciation to Jane Kimbrough for her 
encouragement, support, and thoughtfullness . I also thank 
Dr. M. M. Miyamoto, who generously provided many helpful 
discussions, guidance and much encouragement. Although with 
a very busy schedule, his door was always open for 
discussion and advice on scientific problems. I would like 
to extend appreciation to the other members of my committee, 
Drs . E. Hiebert, G. Moore, and D. Pring who have also 
greatly assisted in my project and have improved the quality 
of the dissertation with their comments. Sincere 
appreciation is also extended to Dr. Hiebert who provided 
laboratory space and supplies for this research. I also 
greatly appreciate the willingness of Dr. R. E. Stall to 
read my dissertation and to participate in my final 

ii 



examination. I have always felt lucky to have such a fine 
committee. I wish to express my deepest thanks to my 
department chairman Dr. G. N. Agrios and Dean J. Fry for 
their generous support and encouragement. The valuable help 
of Drs . F. Martin, S. Pappu and H. Pappu, E. Almira, B. 
Zettler, D. Purcifull, and John Taylor is greatly 
appreciated. I also would like to thank Dr. Gerry and Ulla 
Benny for their assistance and friendship. 

This study could not have been conducted without the 
support of many others. I would like to acknowledge 
Colette Jacono, Rick Smith, Gail Wisler, Rose Koenig, Caryle 
Baker, Gary Marlow, Li-tzu Li, and E. A. Meyer for their 
sincere friendship. I would like to express my heartfelt 
thanks to Emine and Hasan Incirlioglu, Isin and Temel 
Buyuklimanli, Nimet Turel, Sevgi and Dursun Ince, and 
Hayriye and Turgay Ibrikci for their friendship and support, 
and for sharing the good and bad moments with me. Very 
special thanks go to my dearest friend Zekiye Onsan who has 
put up with me for the last ten years. She was always there 
when I needed her. Very special thanks are also due to my 
close friends Nihal and Philip Scarpace for their valuable 
friendship, support, encouragement, and help. 

I would like to express my deepest gratitude to my 
parents, Ayten and Mustafa, my brother Bahadir and his wife 
Zerhan, and my in-laws Sabahat and Rasim for their love, 



111 



support, encouragement, and unlimited understanding. 

To my husband Timur and our daughter, Gulengul go my 
deepest love and gratitude for their love, understanding, 
continuous encouragement, and support during all phases of 
this research. They have shared their love, their lives, 
and their dreams with me. It was through their help and 
love that I found strength to cope with the difficulties 
along the way and accomplish my goal. I do not have enough 
words to thank them. 



IV 



TABLE OF CONTENTS 

page 

ACKNOWLEGMENTS ii 

LIST OF FIGURES vi 

LIST OF TABLES vii 

ABSTRACT viii 

CHAPTER I . INTRODUCTION 1 

The 5S Ribosomal RNA Genes 10 

The 5 . 8S rRNA Genes 12 

The 18S and 28S Genes 13 

The Nonconserved Region of rRNA Genes 15 

CHAPTER II . MATERIALS AND METHODS 19 

Total DNA Extraction 20 

PCR Amplification of rDNA 22 

Sequencing 23 

CHAPTER III . RESULTS FROM MOLECULAR DATA 26 

PCR Amplification 26 

Sequence Alignment 33 

CHAPTER IV. CLADISTICS: MORPHOLOGICAL AND 

ULTRASTRUCTURAL DATA 47 

Explanation for Character States 

Used in This Study 58 

Results of Morphological and 
Ultrastructural Analysis 69 

CHAPTER V. GENERAL DISCUSSION 72 

LITERATURE CITED 81 

BIOGRAPHICAL SKETCH 90 



LIST OF FIGURES 
Figure Page 



1. PCR amplification products of the 5.8S 

and ITS1 of rDNA 27 

2. PCR amplification products of the 5.8S 

and ITS2 of rDNA 28 

3. Percent G+C composition of rDNA regions 32 

4. Multiple sequence alignment of the 

5 . 8S region of rDNA 34 

5. Transversion and transition frequencies 

of species against Neurospora crassa 37 

6. Multiple sequence alignment of the ITS2 
region of rDNA 44 

7. The most parsimonious tree from 

the 5 . 8s sequences 45 

8. The most parsimonious tree from the 

ITS2 sequences 46 

9. Septal structures of various families 

of Pezizales 64 

10. The most parsimonious tree from 
morphological and ultrastructural data 71 



VI 



LIST OF TABLES 



Table page 



1 . Sizes of rDNA Regions 29 

2. Percent divergence values in the 5.8S rDNA 

Out group= Neurospora crassa 40 

3. Percent divergence values in the 5.8S rDNA 

Out group= Saccharomyces carlbergensis 41 

4. Character and Character States of Epigeous 

and Hypogeous Pezizales 54 



vii 



Abstract of Dissertation Presented to the Graduate School of 
the University of Florida in Partial Fulfillment of the 
Requirements for the Degree of Doctor of Philosophy 

NUCLEAR RIBOSOMAL DNA SEQUENCE ANALYSIS IN 
MOLECULAR SYSTEMATICS OF PEZIZALES ( ASCOMYCETES) 

By 

Esengul A. Momol 

August 1992 

Chairperson: J. W. Kimbrough 
Major Department: Plant Pathology 

The Ascomycete order Pezizales is characterized by 
operculate asci borne in an apothecium. Earlier systems of 
classification were based on morphological features of 
ascocarp, asci and ascospores. Recently, ultrastructural 
features of Pezizales have become important in 
classification. Despite the large number of studies, there 
is still considerable uncertainty in the group. There are 
two problems relative to the systematics and phylogeny of 
Pezizales: 1) the limits of families and the number of 
families that should be included in the order and 2) the 
relationship of Pezizales to the other orders of 
Ascomycetes . 

The purpose of this study was to investigate the 
relationships of species within the families, to analyze 
the phylogenetic relationships of families in Pezizales and 
with other orders of Discomycetes, and to investigate the 

viii 



congruence of molecular findings with ultrastructural and 
morphological data. Polymerase chain reaction (PCR) 
amplified 5.8S, ITS1 and ITS2 regions of nuclear ribosomal 
DNA were used for analyzing the phylogenetic relationships 
in this group. Although, the 5.8S coding region was 158bp 
and conserved among the species tested, ITSl and ITS2 
regions were variable in primary size and in sequences. 
Phylogenetic inferences were made by using parsimony 
analysis. Three major groups were found by using the 
sequences from the 5.8S conserved region; however, the 
positions of eight species which belong to the different 
families were resolved by using sequences from the ITS 
region. Parsimony from morphological and ultrastuctural 
data were congruent with the parsimony analysis from 
sequence data. 



ix 



CHAPTER I 
INTRODUCTION 

The Pezizales is a large order of Discomycetes, many 
having cup shaped apothecia and placed in the class 
Ascomycetes . Apothecial shape, however, may be highly 
variable but in most, cylindric asci are arranged in a 
hymenium among sterile paraphyses . Pliny (23-79 A.D.)/ a 
Roman scholar, was the first one to describe the 
Discomycetes as "Pezica", as a kind of mushroom. In 1801, 
Persoon attempted the first systematic classification of the 
Discomycetes. His classification was based on the variation 
of ascocarps. Fries (1822) made a systematic arrangement of 
the Discomycetes according to hymenial configuration. The 
Friesian system of classification remained in use for almost 
60 years. In 1849, Fries modified his system by including 
in the Discomycetes six groups, the Helvellaceae, 
Bulgariaceae, Dermateae, Patellariaceae, Phacidiaceae, and 
Sticteae, based on the microscopic characters of apothecia. 
Although a number of mycologists followed the Friesian 
system, some of them used the different characters of asci 
and ascocarp for the taxonomy of the Pezizales. Crouan and 
Crouan (1857) were the first to demonstrate that 



2 
Discomycetes had both operculate and inoperculate dehiscence 
of asci. 

As research on Discomycetes advanced, microscopic and 
cytochemical features were used more extensively. Nylander 
(1869) used the iodine reaction in asci, Karsten (1869) 
recognized a number of families based on the cellular nature 
of sterile elements of the apothecium, and Saccardo (1884), 
used the size, shape, and septation of ascospores for 
classification. Boudier (1885) was the first to emphasize 
in his new natural classification of Discomycetes the 
presence and absence of an operculum and thus divided the 
group into operculate and inoperculate Discomycetes . In 
addition to ascal dehiscence, he included microscopic 
characteristics such as amyloidity of asci and number of oil 
drops of spores in his classification. He recognized two 
families, Morchelles and Helvelles, with stipitate apothecia 
and alveolate or veined hymenia; two families, Rhizines and 
Pezizes with cupulate, sessile to short stipitate apothecia, 
and three families, Ciliaries, Humaries, and Ascoboles, with 
small, lenticular apothecia. Boudier (1907) modified his 
earlier system of classification by including 7 operculate 
and 12 inoperculate families. He used not only external 
features of apothecia but also cytochemical and microscopic 
observations for delimiting the families. Boudier 's 
contemporaries such as Saccardo (1889), Gaumann (1926), and 



3 
Seaver (1928), however, did not make the sharp distinction 
based on ascal dehiscence but placed both inoperculate and 
operculate taxa together. Nannfeldt (1932) appears to be 
the first to restrict the Pezizales to those taxa with 
operculate asci. He recognized the operculate order 
Pezizales and inoperculate orders Helotiales, Lecanorales, 
and Ostropales in the Discomycetes based on the 
characteristics of spore, asci, and sterile structures. 

In the 1940s, Discomycetes, including Pezizales, were 
reevaluated. Chadefaud (1946) described the internal apical 
apparatus of operculate and inoperculate asci in detail. Le 
Gal (1946) proposed the taxon "subopercules ' for the groups 
having a type of ascal dehiscence intermediate to the 
operculates and inoperculates . She proposed two new names, 
Homospermales and Heterospermales, to replace the classical 
orders Pezizales and Helotiales and those with suboperculate 
asci were placed in the Sarcoscyphaceae. With minor 
modifications, she continued to recognize the families of 
Pezizales previously proposed by Boudier (1907). Korf 
(1954) revised the classification of the operculate 
Discomycetes and proposed 3 families, the Cyttariaceae, 
Pezizaceae, and Sarcoscyphaceae. 

The decade of the 1960s brought about a number of 
changes in the systematics of the Pezizales. Berthet (1964) 
investigated the nuclear condition of spores and other 



4 
apothecial cells. He found that with a few exceptions 
spores of Pezizales were uninucleate. Two notable 
exceptions were the Helvellaceae with tetranucleate and 
Morchellaceae with multinucleate spores. Brummelen (1967) 
described apothecial development and anatomy and proposed 
the term cleistohymenial for those taxa in which the 
ascogenous system is initially closed within the excipulum, 
and gymnohymenial for those in which the asci are exposed 
throughout development. Kimbrough and Korf (1967) examined 
important characters such as microchemical reactions of 
asci, the development of asci and ascospores, and the manner 
of ascal dehiscence in a variety of genera. Rifai (1968), 
using basically morphological and anatomical features, 
followed for the most part the system of classification of 
Le Gal (1953), however, recognizing in addition the family 
Pyronemataceae. Rifai' s most notable proposal was to divide 
the Pezizales into two suborders, the Sarcoscyphineae with 
suboperculate asci and the Pezizineae with regular opercula. 
Kimbrough (1989), using apothecial ontogeny, ascal 
structure, and septal organelles, proposed to recognize the 
Pyronemataceae as a third suborder, the Pyronemineae. In 
1968, Eckblad divided the order Pezizales into the following 
families: Thelebolaceae, Ascobolaceae, Rhizinaceae, 
Pyronemataceae, Helvellaceae, Morchellaceae, Otidiaceae, and 
Sarcoscyphaceae. The predominant feature of his system was 



5 
placing the genera of Humariaceae into two separate 
families, Pyronemateceae and Otideaceae. He also pulled the 
Rhizinaceae from out of the Helvellaceae. 

One of the most widely accepted systems of 
classification of Discomycetes is in Dennis's (1968) 
"British Ascomycetes" which includes the Pezizales, 
Helotiales, Phacidiales, Lecanorales, and Ostropales. In 
dividing the Pezizales, he followed Le Gal's (1953) 
classification with the addition of the Thelebolaceae and 
Aleuriaceae. Arpin (1968) made his classification based on 
the carotenoid content of the spores. These treatments not 
only increase in the number of families of Pezizales but 
also resulted in reclassification of genera within the 
families . 

In the 1970s, ultrastructural studies began to impact 
the systematics of Pezizales. The septal pore structures of 
asci, ascogenous hyphae, and paraphyses have gained lots of 
attention. The septal structures of some families, such as 
the Pezizaceae (Curry and Kimbrough, 1983), Ascobolaceae 
(Kimbrough and Curry, 1985), Ascodesmidaceae (Steffins and 
Jones, 1983), Helvellaceae (Kimbrough, 1989), and 
Morchellaceae (Kimbrough, 1990), are unique and consistent 
at the family level. However, in other families such as 
Pyronemateceae (Kimbrough and Curry, 1986), and 
Thelebolaceae (Kimbrough, 1981), there are complex septal 



6 
structures (Kimbrough, 1986). Merkus (1973) did an 
extensive study on spore ontogeny and she concluded that 
spore ontogeny may not be an appropriate character for the 
taxonomy of Pezizales since different types of spore 
ontogeny may occur in the same genus. However, Dyby and 
Kimbrough (1987) showed that the type of ascosporogenesis 
was consistent in the family Pezizaceae and a similiar 
observation was made by Gibson and Kimbrough (1988) and 
Kimbrough, Wu, and Gibson (1990) in the Helvellaceae, and Wu 
and Kimbrough (1992) in Humariaceae. 

Phylogenetic relationships of the Pezizales have been 
very uncertain over the years . The operculate and 
inoperculate Discomycetes have been considered to be closely 
related because of apothecial shape and hymenial 
configurations. Chadefaud (1946) and LeGal (1946) suggested 
that the Pezizales may have evolved from the Helotiales, 
inoperculate Discomycetes, by way of the Sarcosyphaceae 
(suboperculates) with an intermediate ascal type. Their 
idea was based on the fact that suboperculate asci were also 
found within some inoperculate genera. Many mycologists 
proposed that particular Plectomycetes may be directly 
related to the Pezizales (Malloch, 1979; Benny and 
Kimbrough, 1980). Because of the size and anatomy of 
apothecia, mycologists generally agree that Pezizales are 
closely related to the order Tuberales. The orders have 



7 
usually been separated by the epigeous versus hypogeous 
habitat and the presence or absence of forcible spore 
discharge. Trappe (1979) proposed the reorganization of 
hypogenous Ascomycetes , excluding Elaphomyces / in which the 
Tuberales were included in the Pezizales. The taxonomic 
position of Elaphomyces is not stable. Elaphomyces , having a 
thick peridium and evanescent asci, was thought to be 
distinct from other Tuberales. Korf (1973) placed 
Elaphomyces in a separate family within the Tuberales, but 
at the same time he also noted that it may belong to the 
Eurotiales. 

Controversy also exists relative to the position of 
various genera within the families. Eckblad (1968) placed a 
number of Helvellaceous genera in a new family Rhizinaceae 
based on spore ornamentation. However, current 
ultrastructural studies of ascospores by Gibson and 
Kimbrough (1987) have argued against recognition of the 
Rhizinaceae. A wide difference of opinion exists as to the 
limits of the family Ascobolaceae. Ascobolus , Saccobolus , 
Iodophanus , and Thecothus were placed in Ascobolaceae based 
on morphological studies (Korf, 1973). But ultrastructural 
data from Kimbrough and Curry (1985) showed that the septal 
structure of Iodophanus differs greatly from that of other 
Ascobolaceous genera. They placed Iodophanus in the 
Pezizaceae based on their septal structure similiarity to 



8 
the Pezizaceous genera (Curry and Kimbrough, 1983). Despite 
these extensive studies, there is disagreement as to the 
position of certain genera in almost every currently 
recognized family of Pezizales. 

During the last few decades there has been a growing 
interest among mycologists to reconstruct phylogenetic 
relationships among the fungi by using molecular methods 
(Jahnke, 1987). Many reliable molecular methods are 
available which allow the classification of fungi at 
different taxonomic levels. Nuclear and mitochondrial DNA 
analysis, the molar percentage of DNA bases cytosine and 
guanine, DNA-DNA reassociation experiments, and ribosomal 
RNA (rRNA) seguencing have been utilized. The usefulness of 
mitochondrial and nuclear DNA restriction analysis for 
taxonomic purposes has been repeatedly demonstrated 
(Kozlowski and Stepien, 1982; Kurtzman, 1985). The smaller 
genome size and the faster evolutionary rate make 
mitochondrial DNA more suitable for investigating the 
phylogenetic relationships among the fungi . The multicopy 
nature of the mitochondrial genome and difference in base 
composition from nuclear DNA make it a very valuable 
taxonomic tool especially in yeast systematics (Clark-Walker 
et al . , 1987). DNA-DNA reassociation techniques are based 
on the ability of single stranded DNA molecules to recognize 
complementary single stranded DNA molecules and to form 



9 

duplexes with them in vitro. The data obtained from DNA-DNA 
reassociation experiments depend very much on the kind of 
DNA used. Vilgalys and Johnson (1987) found very similiar 
DNA homology values of repeated DNA and nonrepeated DNA of 
Collybia species, as did Williams and coworkers (1981) for 
Neurospora . Therefore, these data suggest that DNA-DNA 
reasssociation values alone cannot be used as a good 
taxonomic tool. 

Recently, ribosomal RNA genes have proven very 
informative for phylogenetic studies (Jahnke, 1987). 
Ribosomal rRNAs as a taxonomic tool show several advantages 
over other molecular markers, such as being ubiguitous, 
playing a central role in protein synthesis, being highly 
expressed and easily purified. The overall organization of 
rRNA repeat units in higher eukaryotes is in the form of 
long tandem arrays in a head to tail configuration. This 
was first visualized by electron microscopic analysis. 
Synthesis of three RNA molecules found in ribosomes is 
generally achieved via the production of a single rRNA 
precursor molecule. In prokaryotes, the precursor contains 
16S, 23S, and 5S of rRNA. The prokaryotic pre-rRNA 
sometimes contains one or more tRNA molecules. In contrast, 
a eukaryotic pre-rRNA molecule includes a 5 . 8S rRNA and does 
not contain 5S rRNA. In prokaryotic systems, rRNA genes 
exist as few copies, whereas eukaryotic cells contain many 



10 
copies of them. Especially in yeast and Neurospora more 
than 100 copies of rRNA genes are repeated in a tandem array 
(Dutta et al., 1983). The rRNA genes consist of conserved 
regions also called coding regions which include 5S, 5.8S, 
18S, and 28S, and a nonconserved or noncoding regions which 
consists of nontranscribed spacer (NTS), internal 
transcribed spacer (ITS), and external transcribed spacer 
(ETS) regions. 

The 5S Ribosomal RNA Genes 

Of the different ribosomal RNAs, the 5S rRNA was the 
first to be studied extensively, since its length of 120 
nucleotides and makes it easy to seguence. Selker and his 
coworkers (1985) reported on a heterogeneity of 5S rRNAs in 
fungal ribosomes. They showed that six minor kinds of 5s 
rRNA genes exist in Neurospora crassa . 

The rRNA genes function in a folded state by base 
pairing within the molecule to form secondary structures . 
The secondary structure of the 5S rRNA is highly conserved 
in all organisms and consists of 5 stems and 5 loop regions. 
In a majority of basidiomyceteous fungi, 5S rRNA sequences 
fit the general secondary structure model. The 5S ribosomal 
RNA is widely used in Basidiomycetes for phylogenetic 

comparisons . 

■ 



11 

Septal pore structure has been shown to be a valuable 
tool in the systematica of Basidiomycetes (Moore, 1978; Khan 
and Kimbrough, 1982). Walker and Doolitle (1982) using 5S 
rRNA sequences grouped the Basidiomycetes analyzed so far 
into five clusters. They observed that all species 
belonging to clusters 1 and 2 had simple septal pores, 
whereas those belonging to clusters 3 to 5 (except for 
Exobasidium vaccini) had dolipores. These results 
contradict the studies based on morphological characters and 
do not support the idea that the Hetero- and Homo- 
basidiomycetes are two distinct groups within the 
Basidiomycetes. According to 5S rRNA sequence similarity, 
Taphrina deformans is more similiar to Basidiomycetes than 
Ascomycetes (Blanz and Unseld, 1986). It was hypothesized 
that Taphrina may have originated from a primitive ancestor 
of the Ascomycetes or Basidiomycetes. 

In the analysis of additional Basidiomycetes, 
Gottschalk and Blanz (1984) have shown that the 5S rRNA 
sequences have a limited value for differentiation within 
the groups. Mao and his workers (1982) compared the 
sequences of 5S rRNA from Schizosaccharomyces pombe with the 
other species of yeasts, S. cerevisiae and Torula utilis, 
and with the fruitfly Drosophila melanogaster . They 
observed that the sequences in 5S rRNA from S. pombe shared 
more homology to D. melanogaster than to S. cerevisiae and 



12 

T. utilis . All of these results indicate the informative 
limitation of 5S rRNA sequences in evaluation of 
phylogenetic relationships. 

The 5.8S rRNA Genes 

In large subunits of eukaryotic ribosomes, the 5.8S 
rRNA is a specifically bound large subunit rRNA (L rRNA) by 
noncovalent interactions. The 5.8S rRNA-LrRNA complex may 
be isolated from ribosomes in denaturating conditions. In 
the 1980s, several researchers (Jacq, 1981 and Nazar, 1980) 
demonstrated that 5.8S rRNA is homologous with the 5' end of 
prokaryotic 2 3S rRNA. Perhaps, in the course of evolution, 
the 5.8S rRNA originated from the 5' end sequence of 
prokaryotic LrRNA as a result of incorporation of a 
noncoding sequence. In the rRNA operon this sequence is 
represented by the internal transcribed spacer region (ITS)- 
2 between the 3' end of 5.8S rRNA and the 5' end of LrRNA. 
Comparisons of the nucleotide sequences of 5.8S rRNA from 
various sources also indicate a stronger conservation of the 
5' end of the molecule. The degree of homology between 
Neurospora and several species of vertebrates is about 50% 
(Crouch and Bachellerie, 1986). Most of the 5.8S rRNA 
molecules consist of 156 to 167 nucleotide residues. 
Variation in length comes from insertions and the presence 



13 
or absence of additional nucleotide residues at the 5' or 3 ' 
ends. In some species, the 5.8S rRNA molecule is found to 
be heterogeneous at the end. In the yeast 

Schizosaccharomyces pombe , there are 8 strains in which the 
5.8S rRNA is 158 to 165 nucleotide residues long. 

The 5.8S rRNA like other RNAs carries out its function 
in the folded state. There are few models proposed for the 
secondary structures of 5 . 8S rRNA but none of them are in 
full agreement with all the data obtained. The large 
conservation of 5.8S rRNA can be seen among the taxa. The 
identity between compared regions of rodents and man is 100% 
(Nazar et al . , 1976). Alignment of the known 5.8S sequences 
from fungi (S. cerevisiae , N. crassa, and S. pombe), and 
higher eukaryotes shows a high degree of homology (Schaak et 
al.,1982). The comparisons of 5.8S rRNA nucleotide 
sequences may help in determining the degree of affinity 
between the higher taxa such as classes and divisions. 

The 18S and 28S rRNA Genes 

Nuclear sequences for the small (18S) and large subunit 
(28S) ribosomal RNAs are present in the genomes of all 
eukaryotic organisms and have also been used for 
phylogenetic comparisons (Sogin, 1991). The small subunit 



14 
is about 1800 base pairs (bp) and the large subunit is about 
3000bp. 

One major concern when using the structural RNA 
sequences is the affect of substitutions on stem regions. 
The nucleotide change in one strand of the stem region may 
require the a compensatory change in the other strand. The 
18S and 28S rRNAs contain a significant number of 
independently variable sites or divergent domains which may 
be subject to different functional constraints. All 
organisms other than fungi reveal several longer insertions 
in these regions. Nucleotide sequences of domains II and 
III of 28S rRNA which consist of about 1000 nucleotides have 
been determined in species of Saccharomyces , Taphrina, 
Septobasidium , Ustilago, and Exobasidium (Blanz and Unseld, 
1987). The 3' ends of 18S and 28S rRNA genes are highly 
conserved and sequence homology has been found between 
prokaryotes and eukaryotes in these regions . Two complete 
sequences of maize and rice 18S genes showed 97% homology to 
each other (Messing et al. f 1984; Takaiwa et al.,1984). 
Sequence comparisons of the entire 18S genes of Xenopus and 
Saccharomyces indicate extensive but interrupted areas of 
homology (Salim and Maden, 1981). Overall, the 18S gene is 
found to be more conserved than 2 8S gene. 



15 
The Nonconserved Region of rRNA Genes 

The overall structure of the rRNA is described by a 
spacer region and a region giving rise to a primary 
precursor transcript. The precursor rRNA consists of an 
external transcribed spacer (ETS) preceding the 18S RNA gene 
at the 5' end of the transcript, a 5.8S rRNA gene separated 
from both the 18S and 28S rRNA by internal transcribed 
spacers (ITS1 and ITS2), and the 28S rRNA gene at the 3' end 
of the transcript. The most extensive data base for 
interspecific relationships at the rDNA locus comes from 
systematic comparisons of variation in the spacer. The 
large differences in the length of the rRNA repeat unit 
among the eukaryotes are mostly accounted for by variations 
in the size of the nontranscribed spacer regions (NTS) . 
These comparisons have been based primarily on restriction 
enzyme analyses. Most evolutionary studies have confirmed 
the idea that the spacer region is the fastest evolving 
component of the rDNA locus (Crouch and Bachellerie, 1986). 
The ITSs are nonrepetitive and the two spacers (ITS1 and 
ITS2) are not related in sequence. Unlike mature rRNAs, no 
vertebrate ITS sequences show any significant homology when 
compared to yeast (Michot et al., 1982). Much more 
significant homologies in ITS sequences are observed when 



16 
comparing closely related species, such as mouse and rat 
(Michot et al . , 1983). Highly conserved sequences are 
shared between species, and these conserved sequences are 
interspersed with and laterally displaced by highly 
divergent sequences. Furlong and Maden (1983) suggested 
that the displacement is the result of deletions, 
insertions, and possibly point mutations. The lengths of 
ITS1 between species are similiar, whereas ITS2 lengths are 
different. For example, in mouse the ITS2 is 1089 bp, while 
in rat it is 765 bp. However, in yeast (S. pombe ) ITS1 is 
about 400 bp, and ITS2 is 290 bp (Shaak et al.,1982), 
whereas in Verticillium ITSl is about 100 bp and ITS2 is 164 
bp (Nazar et al.,1991). The ITSl of S. carlsberqensis is 
363 bp, about two times the 186 nucleotides of ITSl of N. 
crassa . The length of ITS2 of S. carlsbergensis is also 
almost twice that of N. crassa (235 compared to 145). There 
is little homology between ITS regions of S. carlsbergensis 
and N. crassa (Crouch and Bachellerie, 1986). ITSl 
sequences are more homologous (75%) than ITS2 (30%) between 
rat and mouse. In contrast, ITSl (11%) is less homologous 
than ITS2 (36%) in comparisons of the two Xenopus species. 
Base composition of DNA is an important parameter to 
consider in phylogenetic reconstruction (Larson, 1991). 
Base compositional biases exist in variable regions of rRNA 
genes (Bernardi et al., 1988). The coding region of rRNA 



17 
genes usually are more GC rich than noncoding regions. 
However, in Drosophila , the 28S gene is more AT rich. The 
G+C content ranges in the fungal kingdom from about 27% to 
about 65 mol%. G+C content of Ascomycetes species range 
from 28 to 52 moll, whereas Basidiomycetes range from 49 to 
68 mol% (Jahnke, 1987). The ITS1 of S. carlsbergensis is 
35% G+C, and ITS2 is 39% G+C, whereas in Neurospora , the 
ITS1 and 2 are 55% G+C and 58% G+C, respectively (Crouch and 
Bachellerie, 1986). Storck (1972) suggested that fungal 
evolution was associated with the progressive increase in 
G+C content of the DNA. Similiar G+C value, by itself does 
not indicate the a close relationship but dissimiliar G+C 
value may be a useful characteristic to exclude close 
relationship or conspecif icity . 

Because of the absence of a fossil record, evolutionary 
patterns of fungi have been speculative. In spite of large 
numbers of morphological, cytochemical, and ultrastructural 
studies, there is still considerable taxonomic and 
phylogenetic controversy in the order Pezizales. Among the 
various ultrastructural studies, septal pore structure of 
asci and ascogenous hyphae appear to provide the most 
definitive phylogenetic information (Kimbrough, 1990). 
There have been two problems relative to the systematics and 
phylogeny of the Pezizales; first, the limits and the 
numbers of families that should be included in the order and 



18 
second, the relationship of Pezizales to other subclasses of 
Ascomycetes (Kimbrough, personal communication) . In this 
study, molecular methods were used to clarify the taxonomic 
problems that exist in this group. 

The objectives of this research were (1) to investigate 
the relatedness of species within the families of Pezizales, 
(2) to analyze the phylogenetic relationship of families in 
the Pezizales, (3) to attempt to correlate the 
ultrastructural data with molecular analyses. These goals 
were achieved by using molecular techniques such as 
polymerase chain reaction (PCR) and sequencing of the 5.8S 
and internal transcribed spacer (ITS) regions of rDNA. 



CHAPTER II 
MATERIALS AND METHODS 

Species used in this study representing different 
families of Pezizales were obtained either from cultures or 
herbarium materials. 

Fungal cultures (C) and Herbarium specimens (H) : 
FAM: ASCOBOLACEAE 
(C) Saccobolus depauperatus (Berk, and Br.) Hans. (FLAS 

culture collection 106) (SAC) 
FAM: ASCODESMIDACEAE 
(C) Ascodesmis nigricans Van Tiegh. (FLAS culture 

collection 122) (ASN) 
(C) Ascodesmis sphaerospora Obrist. (FLAS culture 

collection 260) (ASD) 
(C) Eleutroascus lectardii (Nicot) Von Arx. (FLAS culture 

collection 300) (ELE) 
FAM: HELVELLACEAE 
(H) Gyromitra montana Harmaja. (Cryptogamic collections of 

Oregon State University 49452) (GYR) 
(H) Helvella lacunosa Afz.:Fr. (Cryptogamic collections of 

Oregon State University 49459) (HEL) 

19 



20 

FAM: PEZIZACEAE 

(C) Iodophanus sp. (FLAS culture collection 327) (IOD) 

(H) Peziza vesiculosa Bull.: St.Amans. (University of 

Florida Herbarium F53697) (PEV) 
(H) Plicaria endocarpoides (Berk.) Rifai. (University of 

Florida Herbarium F54729) (PAC) 
FAM: HUMARIACEAE 
(C) Lamprospora sp. I (FLAS culture collection 346 from 

Muller) (LAM) 
(C) Lamprospora sp. II (FLAS culture collection 343 from 

Udagawa) (LAU) 
(H) Otidea leporina (Fr.) Fckl . (University of Florida 

Herbarium F49001) (OTI) 
FAM: PYRONEMATECEAE 

(C) Pyronema domesticum (Sow.:Fr.) (ATCC 14881) (PYR) 
FAM: THELEBOLACEAE 
(C) Thelebolus sp. (FLAS culture collection IMI 67944) 

(THE) 
FAM: SARCOSOMATACEAE 
(C) Urnula craterium (Schw.) Fr. (ATCC 11067) (URN) 

Total DNA Extraction 

Total DNA was isolated from the fungal cultures as 
described by Lee and Taylor (1989). Cultures were grown in 



21 
a nutrient rich medium (5 g peptone, 2.5 g yeast extract, 5 
g tryptone, and 10 g NaCl in 1L water) . Lyophilized 
mycelium (20 to 60 mg dry) was ground with a mortar and 
pestle and was put in 1.5-ml Eppendorf microcentrifuge tube. 
Lysis buffer (50 mM Tris-HCl, 50 mM EDTA, 3% SDS, and 1% 2- 
mercaptoethanol) was added, and mixture stirred with a 
dissecting needle and incubated at 65 C for an hour. An 
equal amount of phenol-chloroform (1:1) was added, vortexed, 
and centrifuged at 10,000xg for 15 minutes at room 
temperature. The aqueous phase containing the DNA was 
removed and 3 M sodium acetate was added to the aqueous 
phase followed by 0.54 volumes of isopropanol. The DNA was 
pelleted by centrifuging in a microcentrifuge for 2 minutes 
at room temperature and the pellet was washed with 70% 
ethanol, dried at room temperature for 20-30 minutes, and 
resuspended in distilled water. 

DNA extraction from herbarium materials was done using 
the procedure described by Bruns, Fogel, and Taylor (1990). 
To remove any signs of insect damage, external surfaces of 
herbarium specimens were scraped off. The material (5-10 
mg) was ground to a fine powder and placed in 1.5 ml 
microfuge tubes, suspended in 700 ul of 50 mM EDTA/50 mM 
Tris pH 7.5/ 3% SDS, incubated for 1 hour at 65 C, extracted 
once with phenol-chloroform (1:1), and once with chloroform. 
The phenol was previously equilibrated to pH 7.0. DNA was 
precipitated from the aqueous phase by addition of 50 ul of 



22 

3 M ammonium acetate and by 500 ul of isopropanol. After 
incubation at room temperature, the precipitate was 
centrifuged for 2 minutes, and the pellet was washed with 
70% ethanol and dried at room temperature for 20-30 minutes. 
The pellet was resuspended in 50 ul sterile water and was 
diluted 50 or 1000 fold for PCR amplification. 

PCR Amplification of rDNA 

From 100 to 200 ng of double stranded DNA was amplified 
by using a Perkin Elmer Cetus thermal cycler (Perkin Elmer 
Cetus corporation, Emeryville, CA) . The primers ITS3 (5'- 
GCATCGATGAAGAACGCAGC-3 ' ) , ITS4 ( 5 ' -TCCTCCGCTTATTGATATGC-3 ' ) , 
ITS2 ( 5 ' -GCTGCGTTCTTCATCGATGC-3 * ) , and ITS5 ( 5 ' - 
GGAAGTAAAAGTCGTAACAAGG-3' ) were used to amplify the 5.8S, 
ITS1 and ITS2 regions of ribosomal DNA. These primers were 
provided by John Taylor from the University of California in 
Berkeley. Total reaction volume was 100 ul and contained 
the following components: 50 pmoles of each primer, 2.5 
units Tag polymerase, reaction buffer containing 50 mM KC1, 
10 mM Tris and 2 . 5 mM MgCl 2 , and 200 uM of each of the four 
deoxyribonucleotide triphosphates. These reactions were 
subject to 30-35 cycles under the following temperatures: 2 
minutes at 95 C, 30 seconds at 55 C, 2 minutes at 72 C. 
Results of amplification were assayed for 2 ul aliguots by 



23 

gel electrophoresis in a gel composed of 2% NuSieve and 1% 
normal agarose (FMC, Rocland, Maine). Gels were stained for 
15-20 minutes with 0.5 ug/ml ethidium bromide, destained in 
water for 10-15 minutes, and photographed with polaroid 55 
and 57 film. 

Sequencing 

Products of PCR were extracted once with phenol- 
chloroform and precipitated with 3 M ammonium acetate and 3 
volumes of absolute ethanol. After 2 hours at -80 C the 
precipitate was pelleted by 2-minute centrifugation in a 
microcentrifuge, washed once with 70% ethanol and dried 20- 
30 minutes at room temperature. The pellet was resuspended 
in 100 ul sterile water and diluted to 300 ul and excess 
nucleotides and primers were removed by using the Milipore 
Ultrafree-MC filter unit (Milipore Corporation, MA) . The 
aliquot was centrifuged for 2 to 4 minutes at 2000 x g in a 
fixed angle rotor. The filtrate (50-80 ul) was removed to a 
separate tube and washing steps were repeated twice to 
ensure efficient separation of DNA from primers and free 
nucleotides. Sanger's Dideoxy Chain Termination method was 
used for sequencing (Sanger et al., 1977). Sequencing 
reactions were carried out on 7 ul aliquots of concentrated 
samples using each primer, T7 polymerase, and S labelled 
dATP with using the Sequenase kit (U.S Biochemicals , 



24 
Cleveland, Ohio). Template DNA (0.5-1 ug) was annealed to 
50 pmole primer in a 10 ul reaction by heating to 95 C for 4 
minutes and allowed to cool at room temperature for 1 
minute. Extension was carried out on ice for 5 minutes 
using 1 ul [a- 35 S] dATP (SA>1200ci/mmol) , 1 ul of 0.1 M 
dithiothreitol, 2 ul of diluted (1:5) dNTP mix (USB kit), 
and 2 ul diluted Sequenase T7 DNA Polymerase (1:5). A 3.5 
ul sample of this reaction was added to 2.5 ul of each 
termination mix (USB kit) and reaction was incubated for a 
further 5 minutes at 50 C. The reaction was stopped by the 
addition of 4 ul of formamide dye mix. The samples were 
electrophoresed in 6% polyacrylamide, 7 M urea sequencing 
gels at 1700 volts for 1.5-6 hours. Gels were fixed using 
10% ethanol, and 10% glacial acetic acid for 20-30 minutes 
destained with water for 20-30 minutes, dried, and exposed 
to Kodak X-Omat film. 

Also, some of the sequences from a few species were 
obtained by using automated sequencers, and these sequences 
were compared with manual sequences. Automated sequencers 
utilize electrophoresis but in a different way. They 
measure the time it takes for a band to traverse a specified 
distance in the gel. Thus output is presented as detected 
bands showing peaks on the Y axis and time of 
electrophoresis on the X axis. Each peak is identified as 
an A, T, G, or C depending upon the detection sysytem which 



25 
is based on fluorescent tags to label DNA from different 
reactions (Ferl et al., 1991). 



CHAPTER III 
RESULTS FROM MOLECULAR DATA 

PCR Amplification 

Approximately 200-250 ng DNA from cultures was used for 
PCR amplification. Herbarium materials were diluted 50-to 
1000-fold and generally were subjected to 35 cycles of 
amplification. Primers ITS2 and ITS5 yielded variable sizes 
of amplification products (Fig. 1) which include part of 
5.8S and ITS1 region. Using the primers ITS3 and ITS4, 320 
base pair (bp) of amplification products were obtained (Fig. 
2). These amplification products included part of the 5.8S 
coding region and ITS2 region of rDNA. 

The sizes of ITSl, 5.8S, and ITS2 regions obtained from 
sequences of ribosomal DNA are shown in Table 1. Ambiguous 
sequences from a few species are not included in the Table 
1. The size of the 5.8S coding regions for fifteen species 
tested were 158 bp. However, the ITSl and ITS2 regions were 
variable in size. Closely related species such as 
Ascodesmis nigricans , Ascodesmis sphaerospora , Eleuthroascus 
lectardii , and Saccobolus depauperatus have a conserved ITSl 
region consisting of 170 bp, while the size of this region 



26 



27 



Figure 1. PCR Amplification Products of the 5 . 8S and ITS1 of 
rDNA. 






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ASN= Ascodesmis nigricans 
ASD= Ascodesmis sphaerospora 
ELE= Eleuthroascu s lectardll 
SAC= Saccobolus depauperatus 
PAC= Plicaria endocarpoides 
PEV= Peziza vesiculosa 
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THE= Thelobolus sp. 
PYR= Pyronema domesticum 
GYR= Gyromitra montana 
HEL= Helvella lacunosa 
URN= Urnula craterium 
OTI= Otidea leporina 



28 



Figure 2. PCR Amplification Products of the 5. OS and ITS2 of 
rDNA . 



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ASD= Ascodesmis s phaerospora 
ELE= Eleuthroascus lectardli 
SAC= Saccobolus depauperatus 
PAC= Pllcaria endocarpoides 
PEV= Pezlza vesiculosa 
IOD= Iodophanus sp. 
LAM= Lamprospora sp. 
LAU= Lamprospora sp. 
THE= Thelobolus sp. 
PYR= Pyronema domestlcum 
GYR= Gyromitra montana 
HEL= Helvella lacunosa 
URN= Urnula craterium 
OTI= Otidea leporlna 



29 



Table 1. Sizes of rDNA Regions 



SPECIES 


ITSl(bp) 




5.8S(bp) ITS2(bp) 


PEV 




217 






177 


PAC 




201 






158 


ELE 




170 






158 161 


ASN 




170 






158 161 


ASD 




170 






158 161 


SAC 




170 






158 161 


LAU 




166 






158 163 


LAM 




157 






158 160 


THE 




158 






158 149 


OTI 




201 






158 172 


PYR 




179 






158 163 


IOD 




207 






158 185 


GYR 




363 






158 


HEL 




256 






- 


URN 




462 






172 


NEU 




185 






158 145 


VER 




110 






158 164 


see 




362 






157 235 


Neu (Neuros 
(Saccharomy 


pora), Ver (Verticillium) , and Sec 
ces ) sequences were obtained from the 



Genbank Database, 



30 
ranged from 462 bp in Urnula craterium to only 157 bp in 
Thelebolus . The size variation of ITS2 was considerably- 
less than ITS1. Saccharomyces carlbergensis has a longer 
ITS2 region than the others and Iodophanus sp. with 185 bp 
is the longest among the other Pezizales tested. Closely 
related species, such as Saccobolus depauperatus , 
Eleuthroascus lectardii , Ascodesmis nigricans , and 
Ascodesmis sphaerospora , showed a very conserved ITS2 region 
which is 161 bp in size, similiar to observations from ITS1. 

The large DNA segments called "isochores," showing 
biased G+C/A+T compositions, have been reported in the 
genomes of warm-blooded animals (Bernardi et al., 1988) and 
plants (Salinas et al., 1988). Figure 3 shows the percent 
G+C values in the regions of 5.8S, ITS1, and ITS2 . The G+C 
composition values for the 5.8S in the Ascodesmidaceae which 
includes the species Ascodesmis nigricans , A. sphaerospora , 
and Eleuthroascus lectardii , Pyronemateceae which includes 
Pyronema domesticum , Ascobolaceae family which includes 
Saccobolus depauperatus , Otideaceae family which includes 
Otidea leporina , Helvellaceae family which includes 
Gyromitra montana , Humariaceae family which includes 
Lampspora species, Pyronemateceae family which includes 
Pyronema domesticum Thelobolaceae family which includes 
Thelobolaceae , and Pezizaceae family which includes 
Iodophanus species are similar. 



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Sequence Alignment 

The initial crucial step for analyzing nucleotide 
sequence data is to align the sequences against one another. 
Sequence alignment can be done by eye without using any 
alignment algorithm when DNA homology is conserved (Miyamoto 
and Cracraft, 1991). Since the 5.8S coding region is very 
conserved and free of gaps, initial alignment was done by 
eye. Then different computer assisted alignment programs 
from GCG such as "pileup" and "pretty" for multiple 
sequences, and "seqaid" for pairwise comparisons were used 
to search the best alignment (Figure 4). The 5.8S region of 
Saccharomyces cerevisiae was 157 bp and one gap was 
introduced to facilitate the alignment. The 5' end of the 
5.8S coding region did not show any substitutions among the 
species tested. However, the 3 1 end of the sequences was 
not as conserved as the 5' sequences. 

Because rates of change for different substitution 
types may vary between sequence regions, greater 
phylogenetic resolution may be attained by considering 
transition (TS) to transversion (TV) ratios before assigning 
weights for phylogenetic analysis. Transition and 
transversion frequencies of species were calculated against 
Neurospora for the 5 . 8S rDNA region (Figure 5). For many 
species transitions basically represented more than 50% of 



34 



Lam58s 
Pyr58s 
Asn58s 
Ele58s 
Lau58s 
Asd58s 
Sac58s 
Oti58s 
The58s 
Gyr58s 
Pac58s 
Neu58s 
Scc58s 
Ver58s 
Iod58s 



AAACTTTCAA 
AAACTTTCAA 
AAACTTTCAA 
AAACTTTCAA 
AAACTTTCAA 
AAACTTTCAA 
AAACTTTCAA 
AAACTTTCAA 
AAACTTTCAA 
AAACTTTCAA 
AAACTTTCAA 
AAACTTTCAA 
AAACTTTCAA 
AAACTTTTAA 
AAACTTTCAA 



CAACGGATCT 
CAACGGATCT 
CAACGGATCT 
CAACGGATCT 
CAACGGATCT 
CAACGGATCT 
CAACGGATCT 
CAACGGATCT 
CAACGGATCT 
CAACGGATCT 
CAACGGATCT 
CAACGGATCT 
CAACGGATCT 
CAACGGATCT 
CAACGGATCT 



CTTGGTTCTC 
CTTGGTTCTC 
CTTGGTTCTC 
CTTGGTTCTC 
CTTGGTTCTC 
CTTGGTTCTC 
CTTGGTTCTC 
CTTGGTTCTC 
CTTGGTTCTG 
CTTGGTTCCC 
CTTGGTTCTC 
CTTGGTTCTG 
CTTGGTTCTC 
CTTGGCTCTA 
CTAGGCTCTT 



GCATCGATGA 
GCATCGATGA 
GCATCGATGA 
GCATCGATGA 
GCATCGATGA 
GCATCGATGA 
GCATCGATGA 
GCATCGATGA 
GCATCGATGA 
GCATCGATGA 
GCATCGATGA 
GCATCGATGA 
GCATCGATGA 
GCATCGATGA 
GCATCGATGA 



50 
AGAACGCAGC 
AGAACGCAGC 
AGAACGCAGC 
AGAACGCAGC 
AGAACGCAGC 
AGAACGCAGC 
AGAACGCAGC 
AGAACGCAGC 
AGAACGCAGC 
AGAACGCAGC 
AGAACGCAGC 
AGAACGCAGC 
AGAACGCAGC 
AGAACGCAGC 
AGAACGCAGC 



Lam58s 
Pyr58s 
Asn58s 
Ele58s 
Lau58s 
Asd58s 
Sac58s 
Oti58s 
The58s 
Gyr58s 
Pac58a 
Neu58s 
Scc58s 
Ver58s 
Iod58s 



51 

GAAATGCGAT 

GAAATGCGAT 

GAAATGCGAT 

GAAATGCGAT 

GAAATGCGAT 

GAAATGCGAT 

GAAATGCGAT 

GAAAGGCGAT 

GAAATGCGAT 

GAAATGCGAT 

GAAATGCGAT 

GAAATGCGAT 

GAAATGCGAT 

GAAACGCGAT 

GAAATGCGAT 



AAGTAGTGTG 
AAGTAGTGTG 
AAGTAGTGTG 
AAGTAGTGTG 
AAGTAGTGTG 
AAGTAGTGTG 
AAGTAGTGTG 
AAGTAATGTG 
AAGTAATGTG 
AAGTAATGTG 
AGGTAATGTG 
AGGTAATGTG 
ACGTAATGT . 
ATGTAGTGTG 
AAGTAATGTG 



AATTGCAGAA 
AATTGCAGAA 
AATTGCAGAA 
AATTGCAGAA 
AATTGCAGAA 
AATTGTAGAA 
AATTGCAGAA 
AATTGCAGAA 
AATTGCAGAA 
AATTGCAGAA 
AATTGCAGAA 
AATTGCAGAA 
AATTGCAGAA 
AATTGCAGAA 
AATTGCAGAA 



TTCAGTGAAT 
TTCAGTGAAT 
TTCAGTGAAT 
TTCAGTGAAT 
TTCAGTGAAT 
TTCAGTGAAT 
TTCAGTGAAT 
TTCAGTGAAT 
TTCAGTGAAT 
TTCAGTGAAT 
TTCAGTGAAT 
TTCAGTGAAT 
TTCCGTGAAT 
TTCAGTGAAT 
TCTCGTGAAT 



100 
CATCGAATCT 
CATCGAATCT 
CATCGAATCT 
CATCGAATCT 
CATCGAATCT 
CATCGAATCT 
CATCGAATCT 
CATCGAATCT 
CATCGAATCT 
CATCGAATCT 
CATCGAATCT 
CATCGAATCT 
CATCGAATCT 
CATCGAATCT 
CATTGAATCT 



Lam58s 
Pyr58s 
Asn58s 
Ele58s 
Lau58s 
Asd58s 
Sac58s 
Oti58s 
The58s 
Gyr58s 
Pac58s 
Neu58s 
Scc58s 
Ver58s 
Iod58s 



101 

TTGAACGCAC 

TTGAACGCAC 

TTGAACGCAC 

TTGAACGCAC 

TTGAACGCAC 

TTGAACGCAC 

TTGAACGCAC 

TTGAACGCAC 

TTGAACGCAC 

TTGAACGCAC 

TTGAAAGCAC 

TTGAACGCAC 

TTGAACGCAC 

TTGAACGCAC 

TTGAACGCAC 



ATTGCGCCTC 
ATTGCGCCTC 
ATTGCGCCTC 
ATTGCGCCTC 
ATTGCGCCTC 
ATTGCGCCTC 
ATTGAGCCTC 
ATTGCGCCTC 
ATTGCGCCCT 
ATTGCGCCCG 
ATTGTGACCT 
ATTGCGCTCG 
ATTGCGCCCC 
ATGGCGCCTT 
ATTGCGCCCT 



CTGGTATTCC 
CTGGTATTCC 
CTGGTATTCC 
CTGGTATTCC 
CTGGTATTCC 
CTGGTATTCC 
CTGGTATTCC 
CTGGTATTCC 
CTGGTATTCC 
CCTGTATTCC 
CTGGTATTCC 
CCAGTATTCT 
TTGGTATTCC 
CCAGTATCCT 
ATGGTATTCC 



GGGAGGCATG 
GGGAGGCATG 
GGGAGGCATG 
GGGAGGCATG 
GGGAGGCATG 
GGGAGGCATG 
GGGAGGCATG 
GGGAGGCATG 
GGGGGGCATG 
GGAGGGCATG 
GGAGGGCATG 
GGCGAGCATG 
AGGGGGCATG 
GGGAGGCATG 
GTAGGGCATG 



150 
CCTGTTCGAG 
CCTGTTCGAG 
CCTGTTCGAG 
CCTGTTCGAG 
CCTGTTCGAG 
CCTGTTCGAG 
CCTGTTCGAG 
CCTGTTCGAG 
CCTGTTCGAG 
CCTGTTCGAG 
CCTGTTCGAG 
CCTGTTCGAG 
CCTGTTTGAG 
CCTGTCCGAG 
CCTGTCTGAG 



Figure 4. Multiple sequence alignment of the 5.8S 
region of rDNA from various species of 
Ascomycetes. Species abbreviation are in 
materials and methods. 



35 



Figure 4--continued, 



151 



Lam58s 
Pyr58s 
Asn58s 
Ele58s 
Lau58s 
Asd58s 
Sac58s 
Oti58s 
The58s 
Gyr58s 
Pac58s 
Neu58s 
Scc58s 
Ver58s 
Iod58s 



CGTCATTA 
CGTCATTA 
CGTCATCA 
CGTCATCA 
CGTCATCA 
CGTCATCA 
CGTCATCA 
CGTCATGA 
CGTCATTA 
CCTCGTGA 
CGTCAGCT 
CGTCATTT 
CGTCATTT 
CGTCGTTT 
CGTCAGCT 







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38 
the substitutions in the 5.8S coding region of rDNA. 

Neurospora crassa and Saccharomyces carlberqensis were 
used as outgroups to determine sequence similarity in 
pairwise comparisons. The percent divergences were 
determined by either using the transitions + transversions 
or transversions only according to the following formula: 
D= 100 (transitions and/or transversions + gaps) /total 
nucleotides shared. Transitions begin to saturate when they 
represent about 50% of the substitutions, which is the case 
for the 5.8S coding region, then transversions may be given 
additional weight. Table 2 shows the percent divergence 
values using Neurospora crassa as an outgroup. The percent 
divergence value for Iodophanus sp. was higher than other 
Pezizales species tested when both transition and 
transversion values were used. Thelobolus sp. was the least 
divergent species from Neurospora crassa . However, taking a 
consideration of transversion values only, smaller 
divergence values than those with both transitions and 
transversions resulted. Similar results were obtained when 
Saccharomyces carlberqensis was used as an outgroup (Table 
3) . It was found that Pyronema domesticum was 1% and 5% 
divergent from Saccharomyces , by using the transversion and 
transition values respectively. The percent divergence 
value of 10 was found for Iodophanus by using the 



39 

transitions values but only 3% divergence was found by using 
the transversions . 

Since full sequence alignment should be attempted only 
when it is accepted that the sequences are related from one 
end to the other, multiple sequence alignments of the ITS1 
and ITS2 regions were not possible. Because of similarity 
in length, Ascodesmis nigricans , Ascodesmis sphaerospora , 
Eleutheroascus lectardii, Saccobolus pauperatus , Lamprospora 
sp. I and Lamprospora sp. II, Thelebolus sp. and Pyronema 
domesticum sequences from ITS2 regions were aligned by using 
the pileup multiple alignment program (Figure 6). In this 
study, the comparisons of the 5.8S region from these species 
are not sufficient for making phylogenetic inferences, 
because the 5.8S region is too conserved. However, the ITS 
regions which are not as conserved as coding regions may be 
used to analyze phylogenetic relationships in closely 
related species. Since ITS regions were variable in length, 
gaps were introduced to facilitate the alignment. 

The alignment of sequences was formatted for inferring 
phylogenetic trees using the PAUP (Phylogenetic Analysis 
Using Parsimony) computer program of D. L. Swofford (1990). 
Version 3.0 of this program for the Macintosh system was 
used. All characters were treated as unordered entities. A 
heuristic approach with the tree-bisection-reconnection 
(TBR) branch-swapping algorithm was performed. In the first 



40 



Table 2. Percent Divergence Values in the 5.8S rDNA 



SPECIES 


%DIVERGENCE 


%DIVERGENCE 




(TV+TS+GAP) 


( TV+GAP ) 


LAU 


8.0 


2.0 


LAM 


6.0 


1.8 


PYR 


8.0 


2.0 


ASN 


8.0 


2.0 


ASD 


9.0 


2.0 


PAC 


8.0 


3.0 


IOD 


12.0 


4.0 


GYR 


7.0 


3.0 


OTI 


7.0 


3.0 


ELE 


8.0 


2.0 


SAC 


8.0 


3.0 


THE 


5.0 


1.8 



Outqroup = Neurospora crassa 

Percent divergence values were calculated by using 

the formula: D= 100 ( tv+ts+gap) /total nucleotides shared 

or D= 100 (tv+gap) /total nucleotides shared. 



41 



Table 3. Percent Divergence Values in the 5.8S rDNA 



SPECIES 


%DIVERGENCE 


%DIVERGENCE 




(TV+TS+GAP) 


( TV+GAP ) 


LAU 


7.0 


2.0 


LAM 


6.0 


2.0 


PYR 


5.0 


1.0 


ASN 


7.0 


2.0 


ASD 


8.0 


3.0 


PAC 


8.0 


3.0 


IOD 


10.0 


3.0 


GYR 


9.0 


5.0 


OTI 


7.0 


3.0 


ELE 


7.0 


2.0 


SAC 


7.0 


3.0 


THE 


5.0 


3.0 


Outgroup = 


= Saccharomyces 


carlbergensis 



Percent divergence values were calculated by using 
the formula: D= 100 (tv+ts+gap) /total nucleotides 
shared or D= 100 (tv+gap) /total nucleotides shared. 



42 

analysis transitions and transversions were given equal 
weights. The undetermined two base pairs of the 5.8S region 
from Peziza vesiculosa were treated as missing data and 
included in the analysis. One most parsimonious tree with a 
consistency index of 0.714 was obtained by analyzing the 
sequences from the 5.8S region (Figure 7). Three major 
branches were observed with Neurospora crassa (NEU) as the 
outgroup. Iodophanus sp. (IOD) with Peziza vesiculosa (PEV) 
formed a clade with Plicaria endocarpidales . Otidea 
leporina (OTI) and Gyromitra montana (GYR) were in a 
separate branch, whereas Pyronema domesticum (PYR), 
Eleuthoascus lectardii (ELE), Thelebolus sp. (THE), 
Ascodesmis nigricans (ASN) , Ascodesmis sphaerospora (ASD), 
Saccobolus depauperatus (SAC), Lampospora sp.(LAM), and 
Lampospora sp.(LAU) formed a single group whose lower-level 
relationships were not resolved by using the 5.8S coding 
region sequences. In a second analysis using transversions 
which were not as subject to multiple hits as transitions, 
the same relationships were derived for these eight taxa. 
Since the 5.8S coding region was too conserved to resolve 
lower-level relationships of these eight taxa, sequences 
from variable regions such as ITS were used. The ITS2 
region was selected for this analysis because it was 
similiar in length among the species tested. Heuristic 
approach with TBR algoritm was performed. Iodophanus was 



43 
used as an outgroup. A single most parsimonious tree with a 
consistency index value of 0.538 was found (Figure 8). 
Ascodesmis nigricans , Ascodesmis sphaerospora , and 
Saccobolus depauperatus were clustered together. Two 
Lamprospora species with Thelebolus and Eleuthroascus formed 
a separate clade (Figure 8). Pyronema domesticum formed an 
independent line in this network. 



44 





1 








50 


Asdits2 


.AAAACCTCA 


ACCATAATTT 


ATTATGAGTT 


GGTATTGCAT 


TGGACTTCTT 


Asnits2 


. AAAACCTCA 


ACCATAATTT 


ATTATGAGTT 


GGTATTGCAT 


TGGACTTCTT 


Sacits2 


. AAAACCTCA 


ACCATAATTT 


ATTATGAGTT 


GGTATTGCAT 


TGGCCTTCTT 


Eleits2 


AAAACCTCAA 


CCCATAATTT 


ATTATGAGTT 


GGTTCTGCAT 


TGGACTTTAT 


Lamits2 


AAGACCACTC 


AAGCGA.TTT 


TGCTTGGTAT 


TGGAAGAAGA 


G . . CGCCTCT 


Lauits2 


AAGACCACTC 


AAGCGACTCT 


TGCTTGGTAT 


TGGAAGAAGA 


G. .AGCTTCA 


Pyrits2 


•AAACCTCCT 


CAAGCTCTTT 


TGCTTGGTAT 


TGGAAGAAGA 


GGCCGCTTGT 


Theits2 


51 


CCTCAAGCTT 


TGGTGGGTAT 




CATTGCCAGT 
100 


Asdits2 


GTGGGTCCTC 


TGCGAAATTC 


AATGGCGAAG 


AGCCACGCAA 


CCAAAG .... 


Asnits2 


GTGGGTCCTC 


TGCGAAATTC 


AATGGCGAAG 


AGCCACGCAA 


CCAAAG .... 


Sacits2 


GTGGGTCCTC 


TGCGAAATTC 


AATGGCGAAG 


AGCCACGCAA 


CCAAAG 


Eleits2 


ATGGGTCCTT 


GGTGAAATTC 


AATGGCGAAG 


AGCCACGCAG 


CCAAAG .... 


Lamits2 


GGCCCTCCCT 


TCCGAAATTC 


AATGGCGGAA 


AGTCTCACGT 


GCCCCGGCGT 


Lauits2 


GGCCCTCCCT 


TCCGAAATTG 


AATGGCGGAA 


AGTCTCACGT 


GCCCACGCGT 


Pyrits2 


CGGTCTCCCT 


TTCGAAATGC 


AATGGCAGAT 


TGCCTCATGT 


GCCCTGGCGT 


Theits2 


TTCTGGCAGG 
101 


TCTTAAAATC 


AGTGGCGG . . 


TGCCATTTGG 


CTTCAAGCGT 
150 


Asdits2 


CGTAGTATAA 


CTATTTCGTT 


ATGGAAGCGT 


TGGTGCCTCT 


GCCGTAACCC 


Asnits2 


CGTAGTATAA 


CTATTTCGTT 


ATGGAAGCGT 


TGGTGCCTCT 


GCCGTAACCC 


Sacits2 


CGTAGTATAA 


CTATTTCGTT 


ATGGAAGCGT 


TGGTGACTCT 


GCCGTAACCC 


Eleits2 


CGTAGTATAA 


TAATCTCGTT 


ATGGATGTGT 


GGATACCTCT 


GCCGTAA.CC 


Lamits2 


AGTAAG . TTT 


ATCTTTCGCT 


TGGACCCTGA 


GGCGTTCTCG 


CCCTCAAATC 


Lauits2 


AGTAAG.TTT 


ATCTTTCGCT 


TGGACGCTGA 


GCCCTTCTCG 


CCCTCAAATC 


Pyrits2 


AATAAGATTT 


ATCTTTCGCT 


TGTGCATTGG 


GATGATCCCG 


CCGCAAACCC 


The its 2 


AGTAATTCTT 

151 


CTCGCTTTGG 
166 


AGATCCAGGT 


GGT . TACTTG 


CCAATAACCC 


Asdits2 


CCCAATTTCT 


TAGTTT 








Asnits2 


CCCAATTTCT 


TAGGAT 








Sacits2 


CCCAATTTCT 


TAGTTT 








Eleits2 


CCCAATTTCT 


TAGTTT 








Lamits2 


CCCAATACTC 


TAGG . . 








Lauits2 


CCCAACGCAA 


CACTGG 








Pyrits2 


CCAATTTTTT 


CTGG . . 








The its 2 


CCAATTTTTT 


CAGG. . 









Figure 6. Multiple sequence alignment of the ITS2 region 
from species of different families. Species 
abbreviations are in materials and methods. 



45 




Figure 7. The most parsimonious tree for the 5.8S 

sequences using Neurospora as an outgroup. 
Species abbreviations are in materials and 
methods . 



46 




Asdits2 



Asnits2 



Sacits2 



Eleits2 



Pyrits2 



Lamits2 



Lauits2 



Theits2 



lodits2 



Figure 8. The most parsimonious tree for the ITS2 sequences 
using Iodophanus as an outgroup. Abbreviations are 
in materials and methods. 



CHAPTER IV 
CLADISTICS: MORPHOLOGICAL AND ULTRASTRUCTURAL DATA 

Among the Ascomycetes, the operculate Discomycetes are 
unique because the operculum is not found among other 
Ascomycetes or lichens. Pezizales are also unique in that 
they all have non-septate ascospores. The size and shape of 
Pezizales are highly variable, ranging from small globose 
discoid to large saddle shaped or spongy apothecia (Korf, 
1973). The Tuberales and certain Plectomycetes are believed 
to be derived from the Pezizales (Trappe, 1979; Malloch, 
1979). However, without the fossil record or supported 
hypothesis of geneological relationships within the order, 
ideas on the origin of Pezizales are speculative. 
Within the order Pezizales, as many as eighteen families, 
inclusive of the Tuberales are recognized by different 
mycologists (Eriksson and Hawksworth, 1991). However, there 
is considerable controversy as to the limits of genera and 
families in this order. 

The members of Ascobolaceae are small, mostly dung- 
inhabiting, operculate Discomycetes. The concept of the 
family and the number of genera have changed considerebly 
over the years with as few as three (Rifai, 1968) and as 

47 



48 
many as seventeen genera (Le Gal, 1947; Kimbrough, 1970) 
recognized. Boudier (1869) recognized two subgroups," 
Ascobolei genuini" for the pigmented spored genera, and 
"Ascobolei spurii" for hyaline spored genera. Later, most 
of the hyaline-spored species were transferred to 
Thelebolaceae (Kimbrough and Korf, 1967; Eckblad, 1968) with 
the exception of Iodophanus . Brummelen (1967) described the 
two genera, Ascobolus and Saccobolus forming a sharply 
delimited, natural group in Ascobolaceae. He paid special 
attention to the development and the microscopic structures 
of these fungi in connection with the relationships within 
the genera. Using ultrastructural data, Kimbrough and Curry 
(1985) placed Ascobolus , Saccobolus , and Thecotheus in this 
family based on the ascoboloid septal type. Iodophanus was 
found to have septal structure characteristics of Pezizaceae 
and was transferred to the Pezizaceae. 

Ascodesmidaceae are characterized by small, 
gymnohymenial ascocarps. Ascodesmis , on which the family is 
based, has been placed most often in Ascobolaceae 
(Brummelen, 1967). The taxonomic revision of the genus 
Ascodesmis and family Ascodesmidaceae were delimited and 
defined by Brummelen (1981). The genus Eleuthroascus was 
included in the Ascodesmidaceae because of its septal, 
ascal, and ascospore similiarity to Ascodesmis (Brummelen, 
1989). Kimbrough (1989) placed Ascodesmidaceae and 



49 
Pyronemataceae in a new suborder, Pyronemineae, and 
transferred Amaurascus to Ascodesmidaceae. 

There is considerable controversy regarding the natural 
limits of Helvellaceae (Kimbrough et al., 1990). Nannfeldt 
(1937) recognized the similarity of sessile taxa such as 
Piscina and stipitate genera such as Gyromitra . Berthet 
(1964) demonstrated that the tetranucleate condition of 
ascospores, the habitat, and pigmentation of apothecia were 
features of Helvella , Gyromitra , Rhizina , and Piscina . 
Eckblad (1968) included Rhizina , Piscina , and Gyromitra and 
some related genera with tetranucleate ascospores in a 
separate family, the Rhizinaceae, based on spore 
ornamentation. However, recently Kimbrough and Gibson 
(1988) and Kimbrough (1991) have shown that there are 
similarities of septal pore organelles in "discinoid" and 
"gyromitroid" groups. They include two tribes, Gyromitrae 
and Piscinae in the family Helvellaceae. 

Humariaceae is the largest and most confusing family of 
the order Pezizales. Rifai (1968) demonstrated two types of 
development, with the first series of genera having pale or 
brownish apothecia with slender hyphae, and the second 
series have bright colored apothecium with paraphyses, 
usually turn green in iodine. He proposed that one part of 
Humariaceae might have originated from Helvellaceae. He 
basically recognized four tribes, Otideae, Lachneae, 



50 
Ciliarieae, and Aleurieae. Many genera of Humariaceae were 
placed in the large family Pyronemataceae (Korf, 1973), 
Otideaceae (Eckblad, 1968), and Aleuriaceae (Arpin, 1968). 
Recently, ultrastructural studies by Wu and Kimbrough (1991) 
showed that genera such as Aleuria and Leucoschypha (tribe 
Aleuriae) have an "aleurioid" septal type and produce mostly 
rough spores, and possessed a "gradual condensation" type of 
spore ontogeny. They demonstrated that distinct types of 
spore ontogeny always correlate with specific types of 
septal structures. 

Eckblad (1968) defined a new family Otideacae as having 
medium-sized sessile to shortly stipitate apothecia. He 
included within it the humariaceous genera Geopyxis , Otidea , 
Pustularia , Sowerbyella , and Ascosporassis . Using light 
microscope data, Rifai (1968) concluded with Eckblad and 
others that many similiarites existed between "aleurioid" 
and "otideoid" genera. Korf and Zhuang (1991) placed 
Aleuria , Antracobia , Octospora , and Lamprospora in the 
family Otideaceae based on the apothecium and ascospore 
features. Wu and Kimbrough (1991) determined that Otidea , 
Anthracobia , and Octospora , produce mostly smooth 
ascospores, having with an "antracobioid" septal type and 
"gradual condensation" type of spore ontogeny. They found a 
direct correlation between spore ontogeny and septal 
structures for these genera. 



51 
Pezizaceae is the family of Pezizales with mostly 
medium to large-sized, discoid, cupulate apothecia in which 
the asci turn blue in iodine. Asci that turn blue in iodine 
are also found in Ascobolaceae, and therefore taxa such as 
Iodophanus and Thecotheus , have been transferred in and out 
of the two families. Also, the genus Boudiera was placed in 
Pezizaceae by Korf (1973) and in Ascodesmidaceae by Eckblad 
(1968). Curry and Kimbrough (1983) have found that septal 
structures are unique in the Pezizaceae. Dyby and 
Kimbrough (1987) considered Peziza as a representative genus 
of the family when they studied spore ontogeny and the 
chemical nature of spore walls in selected species. Data 
from these studies and that of earlier work from Curry and 
Kimbrough (1983) on septa showed that these features were 
consistent at the family level. 

Following Rifai (1968) and Arpin (1968), Kimbrough 
(1989) restricted the Pyronemateceae to taxa with small 
gymnohymenial apothecia, hyaline spores, and limited 
exipular growth. Eckblad (1968) appears to be the first to 
greatly expand the limits of the Pyronomataceae to include 
eighteen genera. Among these were genera placed in the 
Aleuriaceae (Arpin, 1968), Ascobolaceae (Brummelen, 1967), 
and Humariaceae (Rifai, 1968). Korf (1972) recognized five 
subfamilies and several tribes which correspond to the 
Ascobolaceae, Aleuriaceae, and Humariaceae of Le Gal (1947), 



52 
including the Otideacae and Thelebolaceae of Eckblad (1968), 
and Aleuriaceae of Arpin (1968). A new suborder 
Pyronemineae was proposed by Kimbrough (1989) to include the 
Pyronemataceae (sensu Rifai) with the genera Coprotus and 

Pyronema . 

Most of the taxa placed in the family Thelebolaceae 
originally represented the "Ascobolei spurii" (Boudier, 
1869), and later recognized as Pseudoascobolaceae (Dennis, 
1968). Many genera such as Ascozonus , Caccobius, 
Coprobolus , Coprotus , Lasiobolus , Thelebolus , and 
Trichobolus were initially placed in this family. Kish 
(1974) found that Coprotus was related to the Pyronema . 
Samuelson (1978) concluded that ascal structures in 
Ascozonus were like those of the Aleuria - Otidea complex. 
Samuelson and Kimbrough (1978) concluded that the ascal 
structure of Trichobolus , which was originally placed in 
Thelebolus , was very different from that of Thelebolus . 
They suggested a relationship of Trichobolus to Lasiobolus , 
but a proper family for these genera has not been 
identified. Kimbrough (1981) and Brummelen (1981) have 
shown that asci of Thelebolus are bitunicate and should be 
excluded from the Discomycetes . Subsequent research has 
shown that the Thelebolaceae family is a very heterogeneous 
group (Kimbrough, 1981). 



53 

The family Sarcoscyphaceae was characterized by 
suboperculate asci and leathery to corky apothecia with a 
gelatenous layer in the excipulum. The family was divided 
into two tribes, Sarcoscyphaceae and Urnuleae, by Le Gal 
(1947). Korf (1970) proposed placing taxa of Urnuleae in 
the Sarcosomataceae. However, ultrastructural studies 
showed that the dehiscence zone of the operculum and ascal 
wall structures of Sarcosomateceae differed from the taxa of 
Sarcoscyphaceae (Samuelson et al., 1978; Kimbrough and Li, 
unpublished) . 

Six of the families placed in Pezizales by Eriksson 
and Hawksworth (1991) are hypogeous , the Monoascaceae is a 
Plectomycetes, and three families are questionable 
operculate Discomycetes . In this part of the study, the 
Parsimony analysis based on the morphological and 
ultrastructural characters of selected families of epigeous 
Pezizales are used to examine the intra- and inter-specific 
relationships, and to test congruence between molecular data 
and morphological observations. 

Genera of nine families of Pezizales were selected and 
subjected to a phylogenetic analysis using PAUP 
(Phylogenetic Analysis Using Parsimony), a computer program 
of D. L. Swofford (1990). Version 3 of this program for the 
Macintosh system was used. These genera correspond to those 
used in the molecular studies described earlier (Chapter 



54 
II). Fifty characters were selected and character states 
were polarized by using Neurospora as the outgroup. The 
characters used in this study are summarized in Table 4. 

Table 4. Character and Character States of Epiqeous and 
Hypoqeous Pezizales 

I. APOTHECIA 

1. Development: = cleistohymenial; 1 ■ gymnohymenial . 

2. Shape: = perithecial; 1 = cupulate; 2 = stalked- 
cupulate; 3 = saddle-shaped; 4 = morchelloid. 

3. Size: = <5mm-5cm; 1 = >5cm. 

4. Size: = 2->5cm; 1 = 5mm-0.3mm. 

5. Texture: = fleshy; 1 = leathery to corky or 
gelatinous . 

6. Color: = non-pigmented; 1 = water soluable pigments; 2 
= carotenoids; 3 = oxydized carotenoids. 

7. Hairs: = absent; 1 = present, nonrooted; 2 ■ rooted. 

8. Substrate: = pyrophilic or soil; 1 = cellulosic; 2 = 
coprophilic . 

9. Substrate: = cellulosic, coprophilic or soil; 1 = 
pyrophilic . 

10. Nutrition: ■ saprobic; 1 = mutualistic symbiosis; 2 = 
antagonistic symbiosis. 



55 

Table 4--continued 

II. ASCI 

11. Operculum: = not present; 1 = functional; 2 - 
nonfunctional. 

12. Dehiscence zone: = not present; 1 = differentiation 
of endoascal wall; 2 = differentiation of exoascal wall. 

13. Operculum: = not present; 1 = apical, thin-walled; 2 
= oblique, thickened lid. 

14. Subopercular shoulder: = not present; 1 - present. 

15. Iodine reaction: = positive; 1 = negative. 

16. Number per apothecium: = numerous; 1 = one to ten. 

17. Phototropism: = absent; 1 = present. 

18. Shape: = narrowly cylindric; 1 = saccate; 2 = 
globose . 

19. Septal structures: = torus, V-shaped striation, 
simple, biconvexed bands; 1 = zonate, hemispherical 
dome . 

20. Septal structures: = hemispherical dome or simple, 
biconvexed bands; 1 = matrix with laminated, translucent 
torus; 2 = granular matrix with V-shaped striations. 

III. ASCOSPORES 

21. Symmetry: = ellipsoid; 1 = globose. 

22. Shape: = symmetrical; 1 = asymmetrical. 

23. Color: = hyaline; 1 = yellow-green; 



56 

Table 4--continued 

2 = purple-brown; 3 = black 

24. Chemistry: = noncyanophilic walls; 1 = cyanophilic. 

25. Chemistry: = noncarminophilic nuclei; 1 = 
carminophilic. 

26. Surface: - smooth; 1 = smooth to slightly ornamented; 
2 = highly ornamented. 

27. Wall ontogeny: = direct precipitation; 1 = gradual 
condensation. 

28. Coherence: = free; 1 = fused. 

29. Lipids: = absent; 1 = small lipids; 2 = large lipid 
droplets . 

30. Granular inclusions: = absent; 1 = present. 

31. Nuclear condition: = uninucleate; 1 = tetranucleate; 
2 = multinucleate. 

32. Spores per ascus: = fewer than eight or eight; 1 = 
greater than 8 . 

33. Spores per ascus: = more than eight or eight; 1 = 
fewer than eight. 

34. Spore liberation: = synchronous; 1 = asynchronous. 

IV. PARAPHYSES 

35. Presence: = pseudoparahyses ; 1 = present; 2 = 
absent . 

36. Pigmentation: = hyaline; 1 = pigmented. 



57 

Table 4 --continued 

37. Branching: = simple; 1 = branched. 

38. Shape: = filamentous; 1 ■ clavate; 2 = setose or 
highly modified. 

39. Nuclear condition: = uninucleate; 1 = multinucleate. 

40. Lipid droplets: = absent; 1 = present. 

41. Granular bodies: = absent; 1 ■ present. 

42. Septal occlusions: = lamellate structures absent; 1 = 
lamellate structures present. 

43. Woronin bodies: = globose; 1 = hexagonal; 2 = long, 
rectangular. 

V. EXCIPULUM 

44. Differentiated: = ectal and medullary areas present; 
1 = excipulum of only one tissue type. 

45. Tissue types: = textura intricata; 1 = textura 
prismatica; 2 = texture angularis; 3 = textura globosa. 

46. Gel tissue: = absent ; 1 = present. 

47. Nuclear condition: = uninucleate; 1 = coenocytic. 

48. Septal structure: = lamellate structures absent; 1 = 
lamellate structures present. 

49. Woronin bodies: = globose; 1 = hexagonal; 2 = long, 
rectangular. 



58 

VI. ANAMORPHS 

50. Anamorphs: = absent; 1 = present. 

Explanation for Character-States Used in This Study 

I. APOTHECIA: 

1(1). Development : All presumed Pyrenomycete and 
inoperculate Discomycetes ancestors have a cleistohymenial 
development. Gymnohymenial development is found in a number 
of Pezizales. 

2(2). Shape : Most Pezizales have sessile, cupulate 
apothecia (Korf,1972), a character shared by most 
Helotiales. Stipitate, saddle-shaped, gyrose, and 
morchelloid apothecial characters are major modifications. 

3(3, 4). Size : A large majority of Pezizales apothecia 
are from 2-5 cm in diameter (Korf, 1972), but several form 
robust (over 5cm) or with small (less than 5mm) apothecia. 

4(5). Texture : Pezizales predominantly have fleshy, 
succulent apothecia, but some are leathery to corky, or 
gelatinous . 

5(6). Color : The ability to produce pigments, such as 
carotenoids, is common in many Pezizales. Arpin (1968) 
considers carotenoids to be good phylogenetic tracers 
because species more advanced chemically have carotenoids 
and the most advanced taxa produce oxydized carotenoids. 



59 
Most families of Pezizales are devoid of carotenoids, but 
instead have a variety of water soluable pigments. 

6(7). Hairs : Excipular hairs are special cellular 
modifications for ecological adaptation. Glabrous apothecia 
are common but others have elaborate and complex types of 
hairs. Eckblad (1968) notes that when different hair types 
are present, it is usually correlated with differences in 
several other characters . 

7(8-10). Substrate : An assumption can be made that most 
Pezizales are saprobic but symbiotic taxa, either 
antagonistic or matualistic, are present. Also, Pezizales 
appear basically cellulosic with many pyrophilic, 
coprophilic, and hypogeous species. Hypogeous Pezizales 
(Tuberales), appear to have become mutualistic symbionts of 
epigeous taxa and evolved a hypogeous habitat. 

II. ASCI: 

8(11-14). Operculum : Although Pezizales are 
characterized by opeculate asci, there are many 
modifications of the operculum (Kimbrough, 1972; Korf, 
1973). The broad spectrum of epigeous Pezizales has an 
operculum in which the dehiscence zone results from physical 
and chemical changes in the inner layer of the ascus wall 
(Samuelson, 1975-1978). Those with other types of operculum 
ontogeny, such as the suborder Pyronemineae have been 



60 

discovered (Kimbrough, 1989). Some have a transition in the 
inner wall to form the operculum, some have pronounced 
shoulders below the operculum and the asci may split 
longitudinally instead of cicumscissily (Kimbrough, 1972), 
while others form thick, lens-shaped structures within the 
operculum (suboperculum sensu Le Gal, 1947). 

9(15). Iodine Reaction : Samuelson (1978a) determined 
that the blue reaction in asci was restricted to an outer 
mucilagenous coat on the outside of the ascus. This state 
is shared by many inoperculate Discomycetes and lichens. 

10(16). Number of Asci per Apothecium : If we derive 
Pezizales from inoperculate Discomycetes (Le Gal, 1953; 
Korf, 1958) we must assume that an extensive multiascal 
hymenium is primitive. Evolution went in two directions, 
one in which there is a sharp reduction in the number of 
asci per apothecium. Examples would include special 
adaptation for the coprophilic habitat (Kimbrough and Korf, 
1967) in which there was a selection pressure for organisms 
with a large projectile. A second direction would be a 
great proliferation in the production of asci, resulting in 
extensive gyrose to spongy, undulating hymenium (Korf, 
1958); this is treated under ascocarp shape. Most differ 
from the outgroup in having a larger number of asci. 

11(17). Spore Ejection ; Most epigeous Pezizales have 
asynchronous spore production, maturation and ejection. 



61 
Some asci become phototrophic (i.e. coprophilic taxa, 
Brummelen, 1967) and protrude strongly above the level of 
the hymenium at spore liberation. Others mature and eject 
their spore synchronously (i.e. certain Sarcoscyphaceae, 
Rifai, 1968). In hypogeous taxa there is for the most part 
no forceful spore discharge. 

12(18). Shape : Eckblad (1968), felt that globose, 
saccate, or broadly cylindric asci were primitive. Such 
conditions are also found in hypogeous taxa in which by 
growing within the substrate there is a restriction in 
normal hymenial growth (Korf, 1958; Trappe, 1979). 
Theoretically, those having evolved the most have lost their 
expansive hymenium of cylindric asci interspersed with 
paraphyses and have a restricted growth state with 
compressed and disarranged asci. Saccate asci in 
coprophilic taxa is the result of selection pressure to form 
large projectiles via large, multispored asci (Kimbrough, 
1981) . 

13(19, 20). Septal pore structures : Various types of 
septal structures were found in epigeous Pezizales. The 
Peziza type septum shows the greatest simplicity and is 
somewhat similar to pit plugging in the red algae (Pueschel 
and Cole, 1982). The ontogeny and structure of pore plugs 
in the Florideophyceae is similiar to that of the Pezizaceae 
(Curry and Kimbrough, 1983) in the formation of electron- 



62 
opaque, biconvex bands. In Pezizales, ascal septa appear to 
be in two major lines of evolution, one in which there is 
additional plug deposition onto the convexed or biconvexed 
band to form a zonate, hemispherical dome and the second in 
which there is an electron-translucent torus bordering the 
pore in which there is what appears to be a loose matrix of 
granular material. This material usually accumulates within 
the ascus side of the septal pore. The matrix becomes more 
pronounced and with V-shaped striations in certain 
Humariaceae ( Pulvinula and Geopyxis ) and in members of 
Helvellaceae and Morchellaceae (Kimbrough and Gibson, 1989; 
Kimbrough, 1991). These two patterns of pore plug 
development are outlined in Figure 9. 

III. ASCOSPORES: 

14(21, 22). Shape : Most epigeous and hypogeous 
Pezizales have broadly ellipsoid spores (Korf, 1972; Trappe, 
1979). When Pezizales evolved opercula, an ellipsoid spore 
evolved concurrently to optimize spore ejection. There 
appear to be two lines of evolution, one in which the spores 
become globose, for example in the hypogeous and few 
epigeous taxa, and second, those that become cylindric or 
inequalateral (Sarcoscyphaceous taxa, Rifai, 1968). Several 
deviations from a broadly ellipsoid shape are present within 
the order. 



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15(23). Color : With minor exception, spores of 
Pezizales are hyaline (Korf,1973; Eckblad, 1968). 
Pigmentation of spores in Ascobolaceae (Brummelen, 1967), 
Ascodesmidaceae (Brummelen, 1989), and certain Pezizaceae 
(Korf, 1973) characterize those groups. 

16(24, 25). Chemistry : A number of taxa of Pezizales 
have spores with peculiar staining reactions such as 
carminophily or cyanophily (Rifai, 1968; Eckblad, 1968; 
Korf, 1973), while most of the remainder are unreactive in 
various mounting agents. 

17(26). Spore Surface : A majority of Pezizales have a 
smooth spore surface, a condition shared by most 
inoperculate Discomycetes (Korf, 1972). A large group has 
taxa with smooth to ornamented surfaces, while a smaller 
number have exclusively ornamented spores. 

18(29, 30). Inclusions : The primitive ascophyte (Cain, 
1972) or ancestral Pezizales probably had merely the 
essential cellular organelles, i.e., nuclei, mitochondria, 
and ribosomes. Additional organelles such as large lipid 
droplets (with or without carotenoids ) , polysaccaride 
granules or similiar inclusions occur. DeBary bubble 
formation (a gaseous interface) appears to be a special 
reaction to certain mounting media (Kimbrough nad Korf, 
1967). It is doubtful that it is of any phylogenetic 
significance. 



66 
19(31). Nuclear Condition ; With a few exceptions, 
spores of Pezizales are uninucleate (Berthet, 1964). 
Tetranucleate spores characterize Helvellaceae, and the 
multinucleate condition is a characteristic feature of 
Morchellaceae and is very common in hypogeous taxa. 
Parguey-Leduc and coworkers (1987) have shown that the 
multinucleate condition in species of Tuber is the result of 
atypical spore delimitation by the spore delimiting 
membranes. Tetranucleate spores of Helvellaceae (Gibson and 
Kimbrough, 1988) and multinucleate spores of Morchellaceae 
(Gibson and Kimbrough, 1988) are the result of mitotic 
divisions within the initial uninucleate spore. 

20(32, 3). Number of Spore per Ascus : Eight-spored asci 
are the rule in Ascomycetes. However, there are some taxa 
with fewer or more spores per ascus. Multispored asci are 
most common among coprophilous Discomycetes (Kimbrough and 
Korf, 1967; Brummelen, 1967). Tetra-spored asci are less 
common in Pezizales (Eckblad, 1968; Rifai, 1968; Korf, 
1972), but some eight-spored taxa produce four or fewer 
spores when grown in culture. Greatly reduced spore numbers 
are common in hypogeous Pezizales (Trappe, 1979), and is the 
result of atypical ascosporogenesis . 

IV. PARAPHYSES: 

21(35). Presence or Absence : With minor exceptions, 



67 
asci of Pezizales are interspersed with paraphyses (Korf, 
1973). A number of hypogeous Pezizales have asci that are 
seperated by pseudoparenchyma cells (Gilkey, 1939; Trappe, 
1979) . 

22(36). Pigmentation : A large majority of Pezizales has 
hyaline paraphyses (Kimbrough, 1970; Korf, 1972). But many 
taxa have paraphyses containing carotenoids or other 
pigments . 

23(37). Branching : Most epigeous Pezizales have simple, 
unbranched paraphyses (Korf, 1972). But many have branched 
and anastomosing paraphyses. Hypogeous taxa are 
characterized by having highly branched and anastomosing 
paraphyses (Trappe, 1979). 

24(38). Shape : Filamentous paraphyses characterize most 
Pezizales (Korf, 1972; Trappe, 1979), but broadly clavate or 
moniliform paraphyses occur. 

25(39). Nuclear Condition : Berthet (1964) contends that 
the uninucleate state is more primitive than the coenocytic 
condition. The uninucleate condition is found in 
inoperculate Discomycetes and in a few operculate taxa but 
most operculates are coenocytic. 

26(40, 41). Inclusions : Paraphyses of most Pezizales 
are without inclusion bodies. The formation of lipid 
(without or without caretonoids) , granules, or other 
inclusion bodies are also common. 



68 
27(42, 43). Septal Structures ; All Pezizales (including 
hypogeous taxa) studied thus far have a granular lamellate 
structure within septal pores of paraphyses (Curry and 
Kimbrough, 1983; Kimbrough, 1991). They are considered 
uniguely pezizalean because they are not found in other 
Ascomycetes . Evidence suggests that Woronin bodies are 
composed largely of lipoproteins (Wu and Kimbrough, 1991). 
There appears to be great differences in concentration of 
proteins in Woronin bodies of various taxa of Pezizales, and 
perhaps accounts for morphological variations seen in 
Woronin bodies (Kimbrough, 1991). Simple, globose Woronin 
bodies are most common, but hexagonal, long, and rectangular 
types also occur. 

V. EXCIPULUM: 

28(44). Ectal and Medullary Differentiation : A majority 
of Pezizales has well differentiated ectal and medullary 
excipular layers (Korf, 1972; Gilkey, 1939; Trappe, 1979). 
This trait is not shared with the outgroup, Neurosopora 
crassa . Reduced and highly modified excipula are common 
among Pezizales. 

29(45). Tissue Types : There are several types of 
tissues found within the excipulum of epigeous and hypogeous 
Pezizales (Korf, 1973). It is difficult to project what 
types of tissues were present in ancestral Pezizales, 



69 
although the outgroup, Neurospora , has predominantly a 
texture prismatica to textura angularis. The simplest type 
of tissue consists of interwoven hyphae (textura intricata) , 
but other special arrangements or modifications of this type 
are present. 

30(46). Gel Tissues : A number of Pezizales have 
specialized gel tissues either in the medullary or ectal 
excipulum (Moore, 1965). 

31(47). Nuclear condition : Berthet (1964) and Eckblad 
(1968) considered the uninucleate condition of exipular 
cells to be primitive. Most Pezizales have coenocytic 
excipular cells. 

32(48, 49). Septal Structures : Septal pore structures 
in excipular cells parallel those of the paraphyses . All 
are characterized by the presence of a granular matrix with 
striations, the "lamellate structure" (Curry and Kimbrough, 
1983), that varies somewhat in different taxa. Woronin 
bodies also may be globose, hexagonal, long, or rectangular. 
Globose Woronin bodies are found consistently throughout 
Ascomycetes . 

Results of Morphological and Ultrastructural 
Analysis 

Cladistics using parsimony analysis was performed with 
the PAUP (Phylogenetic Analysis Using Parsimony) software of 



70 
Swafford (1991). A single most parsimonious tree was found 
with a consistency index value of 0.511. 

The two Ascodesmis species clustered together and 
appear to be more closely related to Saccobolus and 
Eleuthroascus than Peziza (Figure 10). Pezizaceous genera, 
Peziza , Plicaria , and Iodophanus , formed a separate branch, 
suggesting a close relationship to the helvellaceous genera 
Helvella and Gyromitra . The two Lamprospora species showed 
a close affinity to Ascodesmidaceae. Otidea (Humariaceae) 
appears to be more closely related to Urnula 
(Sarcosomataceae) than to Pezizaceae. Thelebolus 
(Thelobolaceae) appears not to be closely related to either 
Ascodesmidaceae, Ascobolaceae, Pezizaceae or Humariaceae. 



71 



NEU 



SAC 



ASN 
ASD 



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LAU 



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PEV 
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THE 



Figure 10. The most parsimonious tree in the analysis using 
the morphological and ultrastructural data. 
Abbreviations are in materials and methods. 



CHAPTER V 



GENERAL DISCUSSION 



Fifteen species which belong to different families of 
the order Pezizales were investigated in this study. For a 
more complete analysis and identification of inter-specific 
variation among the species, amplification and direct 
sequencing of nuclear ribosomal DNA were done. 
The primers ITS2, ITS3, ITS4, and ITS5 specifically 
amplified the ITS1, 5.8S, and ITS2 regions of ribosomal DNA. 
In contrast to the size of the 5.8S region which was 
conserved among the species, the size of ITS1 and ITS2 was 
variable. The species which were closely related based on 
morphological characters, such as Ascodesmis nigricans , 
Ascodesmis sphaerospora , Saccobolus depauperatus , and 
Eleuthroascus lectardii , showed a conserved ITS1 region of 
170 bp in size. Recently, DNA sequence comparisons of the 
nuclear ITS region showed that genera of the family 
Sordariaceae (Ascomycetes) are no more divergent in this DNA 
region than are species of Laccaria or Suillus that belong 
to different orders of Basidiomycetes . This cannot be 
easily explained. One thought might be that speciation may 



72 



73 
have occurred more rapidly in Sordariaceae, considering the 
life cycles of saprobic Ascomycetes, than in mycorrhizal 
Basidiomycetes . Within Armillaria species, ITS sequences 
were insufficient to resolve relationships within all 
species; thus the other variable regions of rDNA such as NTS 
(nontranscribed spacer) or IGS (intergenic spacer) might be 
more useful (Bruns et al.,1991). In this study the ITS 
region was very useful for resolving uncertain positions of 
closely related species. The cluster of eight species 
(Figure 8) was resolved by using the variable region ITS2. 

In plants and animals, it has been reported that 
certain DNA regions called isochores show biases of G+C/A+T 
compositions (Salinas et al . , 1988; Bernardi et al., 1988). 
Takaiwa and et al. (1985) showed that ITS1 and ITS2 regions 
of O. sativa have high G+C contents of 72% and 77% 
respectively. However, Yokota et al. (1989) showed that G+C 
contents of D. carota and V. faba ITS1 and ITS2 are 
approximately 50%. The similarity values between these two 
dicots were 49% in the ITSl and 53% in ITS2 regions, in 
contrast to the value of 93% observed between their 5.8S 
rDNA sequences . Both ITS sequences in mouse are very rich 
in G+C, 70% and 74.3% for ITSl and ITS2 respectively. Also 
for each ITS, the number of Gs equals exactly the number of 
Cs (350:350 in ITSl, 407:407 in ITS2). Both ITS are 
particularly deficient in As: 7.1% and Ts : 6.2% for ITSl and 



74 
ITS2 respectively (Furlong and Maden, 1983). In this study, 
similiar percentage G+C values were found in the ITS and 
5.8S regions of Gyromitra , Lamprospora , and Thelebolus , 
Ascodesmis , Eleuthroascus , Saccobolus , Pyronema, and 
Iodophanus (Figure 3). These base composition features 
clearly distinguish ITS sequences from 5.8s rRNA. The data 
obtained from plants support this argument (Torres et al., 
1990). However, discrepancies occur with respect to base 
composition in protozoans such as between Crithidia 
fasciculata and Giardia lambia . 

When mouse ribosomal ITS sequences are compared with 
yeast and Xenopus , a complete lack of homology is observed, 
except for a short segment (13 bp) located immediately 
downstream from the 5.8S rRNA which is conserved between the 
two vertebrates . When Neurospora and Saccharomyces ITS 
sequences are compared very little homology was found. It 
has been observed that the sequences of the ITS region from 
different species of families of Pezizales showed inter- 
specific variation that appears to be more than intra- 
specific variation. According to Michot (1982) the pattern 
of distribution of homologies between the species in areas 
of ITS may have been subject to selective pressure and could 
have some functional roles, probably in the control of 
ribosomal biogenesis. 

Different parts of the RNA molecule may be subject to 
different selection pressures. It has been recently 



75 
suggested that unpaired rRNA regions may give more reliable 
results at distant evolutionary distances than paired 
regions (Wheeler and Honeycutt, 1988). However, the 
available data indicated that the nucleotide sequences of 
5.8S were highly conserved during evolution, but have 
changed to such an extent that phylogenetic trees can be 
constructed among families and orders. By contrast, the 
nontranscribed and transcribed spacer regions of rDNA were 
very variable among different organisms and show high 
sequence heterogeneity. 

Based on morphological data, various evolutionary 
pathways for Pezizales were proposed by many mycologists. 
Atkinson (1915) and Gaumann (1926, 1964) viewed Pezizales as 
evolving from a lower group of Ascomycetes such as yeast- 
like fungi (Endomycetales) . Nannfeldt (1932), LeGal (1953), 
Berthet (1966), and Arpin (1968) all considered the 
Pezizales to have evolved from the inoperculate family 
Sclerotiniaceae. Their ideas were based on the study of 
LeGal (1946) who visualized the suboperculate Discomycetes 
as being an intermediate group between the operculate and 
inoperculate Discomycetes. LeGal (1953) suggested a number 
of phylogenetic lines in the Pezizales which would suggest a 
polyphyletic origin of various families. Along one line she 
projected that members of Otideaceae (=Humariaceae sensu 
LeGal) gave rise to the Pezizaceae (=Aleuriees) and this 



76 
family in turn gave rise to Ascobolaceae. In another line, 
members of the Sclerotiniaceae gave rise to other groups of 
Humariaceae. Gaumann( 1964) viewed the evolution of 
Pezizales from the Plectomycetes, going sequentially from 
small gyimnohymenial taxa such as Pyronema to large complex 
members of Morchellaceae and Helvellaceae. Using karyotype 
data, Berthet (1966) suggested an origin of various families 
of Pezizales from the inoperculate Discomycete order 
Helotiales. Those with uninucleate ascospores and excipular 
cells were lower on the evolutionary chain than 
multinucleate Helvellaceae, Morchellaceae, or 
Sarcoscyphaceae. Eckblad (1968) viewed Pezizales as 
evolving from Pyrenomycetes . He felt that highly reduced 
cleistohymenial members of Thelebolaceae were very similiar 
in structure and ontogeny to perithecial members of the 
Pyrenomycetes. Through the elaboration of ascocarps, 
various other families evolved in which Morchellaceae, and 
Helvellaceae were at the end of the evolutionary chain. 
Cytochemical data such as carotenoids or other pigments led 
Arpin (1968) to agree with LeGal ' s opinion that 
suboperculate Sarcoscyphaceae evolved from inoperculate 
Sclerotiniaceae. He would similarly derive the Otideaceae 
and Pezizaceae from the inoperculate family Geoglossaceae. 
Based on septal structures, Wu (1991) demonstrated that 
there was a strong correlation of septal structures among 



77 
various families of Pezizales. He proposed an evolutionary 
model linking Humariaceae with other families of Pezizales. 
Species producing smooth ascospores and simple apothecia 
such as Pyronema were considered to be primitive, and from 
this group there seems to be two directions of evolution, 
one connecting two families, Pezizaceae and Ascodesmideceae 
then to Ascobolaceae, and the other to both Helvellaceae and 
Morchellaceae. Ascobolaceae is thought to be an advanced 
group because of its complex spore ontogeny. 

In this study, using the 5.8S and ITS sequences, 
phylogenetic inferences were made in various species 
representing different families of Pezizales. The position 
of Plicaria , Iodophanus , and Peziza in the Pezizaceae 
appears to be confirmed and tends to support septal 
ultrastructure data that show these genera to be closely 
related (Kimbrough and Curry, 1985). Although Iodophanus 
has been placed among the Ascobolaceae (Brummelen, 1967; 
Dennis, 1978), the 5.8S and ITS data do not support this 
position. The parsimony analysis of morphological and 
ultrastructural data would suggest a close affinity of 
Pezizaceae and Helvellaceae. There appears to a close 
correlation of results from the parsimony analysis of 
molecular data and that of morhological and ultrastructural 
data. Also, the two Lamprospora species which were 
clustered together in both analyses and appear to be more 



78 

closely related to Ascodesmis than to Iodophanus (Figure 8 
and 10). This disagrees with the most current systematic 
arrangements (Eckblad, 1968; Rifai, 1968; Korf, 1973). It 
has been observed that there seems to be a disagreement 
regarding the position of Pyronema between morphological- 
ultrastructural cladistics analysis and molecular parsimony 
analysis. In the parsimony analysis from molecular data, 
Pyronema shows a close relationship to species of 
Lamprospora , whereas, in cladistics analysis from 
morphological and ultrastructural data Pyronema is more 
closely related to Otidea than Lamprospora . 

For many years the species of Ascodesmis were placed 
within the Ascobolaceae (Brummelen, 1967; Dennis, 1968). 
More recently Ascodesmis has been placed in the family 
Ascodesmidaceae (Brummelen, 1981). Brummelen (1989) and 
Kimbrough (1989) both concluded that based on morphological 
and ultrastructural data Eleuthroascus also belongs to the 
Ascodesmidaceae. All data available from the 5.8S region as 
well as cladistics of morphological and ultrastructural 
data support the recognition of this family and a close 
relationship of Ascodesmis and Eleuthroascus . However, when 
the ITS2 region was analyzed Eleuthroascus was set apart 
from Ascodesmis . In fact it appears to be closely related 
to Pyronema . Also, it appears from these data that the 
Ascodesmidaceae is more closely related to Ascobolaceae than 



79 
to the other families of Pezizales. 

Gyroimitra and other Helvellaceae with elaborate 
ascocarps and unique cytological and ultrastructural 
features are believed to be highly evolved (Berthet, 1964; 
Arpin, 1968; Eckblad, 1968; Gibson and Kimbrough, 1987; 
Kimbrough, 1989). There have always been questions, 
however, as to from which group of sessile, cupulate 
Pezizales they may have evolved. Using the 5.8S sequence 
data, Gyromitra and Otidea appear to be closely related. 

The taxonomic position of Thelebolus species in the 
Pezizales has been controversial for several years 
(Kimbrough, 1981). The research of Samuelson and Kimbrough 
(1978) and later Kimbrough (1981) shows that species of 
Thelebolus belong to the Loculoascomycetes since they have 
bitunicate asci, with the "jack-in-the-box manner" of ascal 
dehiscence. However, Eriksson (1984) does not feel that 
Thelebolus should be removed from Pezizales. But both 
cladistics analysis using ultrastructural and morphological 
data, and the parsimony analysis from 5.8S and ITS sequences 
support the view that Thelebolus is not closely related to 
the species placed in Pezizales. 

Finally, in the comparison of morphological and 
ultrastructural data with molecular information, the 
following conclusions were reached: 
(1) Thelobolus is not closely related to species of 



80 

Pezizales tested. 

(2) A lack of similarity between Ascodesmis 
(Ascodesmidaceae) species and Otidea (Humariaceae) was 
observed. 

(3) Gyromitra (Helvellaceae) is more closely related to 
Otidea (Humariaceae) than Ascodesmidaceae. 

(4) Ascodesmidaceae is more closely related to the 
Ascobolaceae than the other families of Pezizales. 

(5) The parsimony analysis from molecular data does not 
agree with the cladistic analysis from morphological 
and ultrastructural data regarding the position of 

Pyronema . In the parsimony analysis, Pyronema 
(Pyronemateceae) is closely related to the species of 
Lamprospora (Aleurieae sensu Korf, 1973), whereas, in 
the cladistic analysis Pyronema shows a close 
relationship to Otidea (Otideeae sensu Korf, 1973). 

(6) The Lamprospora species examined were more closely 
related to Ascodesmis species than to Iodophanus 
(Pezizaceae) . 

(7) An overall congruence between data from morphology- 
ultrastructural features of Pezizales and data from 
5.8S and ITS sequences exit. However, the 
quantification of congruence needs to be done. 



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BIOGRAPHICAL SKETCH 

Esengul Ayse Momol was born May 15, 1956, in Kutahya, 
Turkey. She received a Bachelor of Science degree from the 
University of Ege, College of Pharmacy, in 1980. She first 
started working in Pharmacology department at the University 
of Florida under the guidance of Dr. E. Meyer, then she 
continued her work as a director of brain cell culture 
facility in Dr. M. Raizada's laboratuary located in 
Physiology Department at the University of Florida. In 
1986, she was admitted to graduate school and in 1989, she 
received her Master of Science degree, specializing in the 
molecular genetics of fungus-plant interaction from Plant 
Pathology Department of the University of Florida. She has 
continued her studies toward the completing a Doctor of 
Philosophy degree in the molecular systematics. 



90 



I certified that I have read this study and that in my 
opinion it confirms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 

James W. Kimbrou<|h, Chairman 
■Professor of Plant Pathology 



I certified that I have read this study and that in my 
opinion it confirms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 





Daryl "Pring J 

Professor of/Plant Pathology 



I certified that I have read this study and that in my 
opinion it confirms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 






^l! £Ub 



Ernest Hiebert 

Professor of Plant Pathology 



I certified that I have read this study and that in my 
opinion it confirms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 



■QvaA^Aw \ tA^x<5<?__£2_ 



Gloria Moore 

Professor of Horticultural 

Science 



I certified that I have read this study and that in my 
opinion it confirms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, as 
a dissertation for the degree of Doctor of Philosophy. 




M. M. Miyamoto 
Associate Professor of 
Zoology 



This dissertation was submitted to the Graduate Faculty 
of the College of Agriculture and to the Graduate School and 
was accepted as partial fulfillment of the requirements for 
the degree of Doctor of Philosophy. 



August, 1992 




ouJz, X- <JO 



Dean, /College of Agriculture 




Dean, Graduate School